DNA polymerase III holoenzyme from Escherichia coli

ABSTRACT

The present invention is directed toward the 5 previously unknown genes, for subunits  delta ,  delta &#39;,  chi ,  theta , and  psi , of the DNA polymerase III holoenzyme, and toward a unique man-made enzyme containing 5, preferably 6, protein subunits which shows the same activity as the naturally occurring 10 protein subunit DNA polymerase III holoenzyme.

Research support which led to the making of the present invention was provided in part by funding from the National Institutes of Health under Grant No. GM-38839. Accordingly, the federal government has certain statutory rights to the invention described herein under 35 U.S.C. 200 et seq.

The present application for Letters Patent is a continuation-in-part of my earlier U.S. patent application Ser. No. 07/826,926, filed Jan. 24th 1992., now abandoned said continuation-in-part having been filed as International Patent Application PCT US93/00627 on Jan. 22nd 1993.

In 1982, Arthur Kornberg was the first to purify DNA polymerase III holoenzyme (holoenzyme) and determine that it was the principal polymerase that replicates the E. coli chromosome.

In common with chromosomal replicases of phages T4 and T7, yeast, Drosophila, mammals and their viruses, the E. coli holoenzyme contains at least ten subunits in all (α, ε, θ, τ, χ, δ, δ', χ, ψ, β) [see J. Biol Chem, 257:11468 (1982)]. It has been proposed that chromosomal replicases may contain a dimeric polymerase in order to replicate both the leading and lagging strands concurrently. Indeed the 1 MDa size of the holoenzyme and apparent equal stoichiometry of its subunits (except β which is twice the abundance of the others) is evidence that the holoenzyme has the following dimeric composition: (αεθ)2τ2(γδδ'χψ)2β4.

One of the features of the holoenzyme which distinguish it as a chromosomal replicase is its use of ATP to form a tight, gel filterable, "initiation complex" on primed DNA. The holoenzyme initiation complex completely replicates a uniquely primed bacteriophage single-strand DNA (ssDNA) genome coated with the ssDNA binding protein (SSB), at a speed of at least 500 nucleotides per second (at 30° C.) without dissociating from an 8.6 kb circular DNA even once. This remarkable processivity (nucleotides polymerized in one template binding event) and catalytic speed is in keeping with the rate of replication fork movement in E. coli (1 kb/second at 37° C.). In comparison, DNA polymerase I as well as the T4 polymerase, Taq polymerase, and T7 polymerase (sequence) are all very slow (10-20 nucleotides) and lack high processivity (10-12 nucleotides per binding event). With these distinctive features the polyIII holoenzyme has commercial application. However, there is a good reason it has not yet been applied commercially. Namely, there are only a few (10-20) molecules of polyIII holoenzyme per cell and thus it is difficult to purify; only a few tenths of a milligram can be obtained from 1000 liters of cells; and it can not be simply overproduced by genetic engineering because it is composed of 10 different subunits.

The subunits of DNA polymerase III holoenzyme are set forth in the following table:

    ______________________________________                                         Gene Subunit Mass (kda)                                                                               Functions                                               ______________________________________                                              α 130       DNA polymerase                                               ε                                                                              27        Proofreading 3'-5' exonuclease                               θ 10                                                                     τ   71        Dimerizes core, DNA-dependent ATPase                         γ 47        Binds ATP                                               holA δ 35        Interact with g to transfer β to DNA               holB δ'                                                                               33        DNA-dependent ATPase with g                             holC χ   15                                                                holD ψ   12                                                                holE β  40        Sliding clamp on DNA, binds core                        ______________________________________                                    

As discovered in making the present invention, the δ' is a mixture of two proteins, both encoded by the same holB gene, and therefore it may be regarded as two subunits of the holoenzyme, thus bringing the total number of subunits in the holoenzyme to eleven.

The genes for 5 of the holoenzyme's subunits have been identified [see Nucleic Acids Research 14(20): 8091 (1986); Gene 28:159 (1984); PNAS (U.S.A.) 80:7137 (1982); J. of Bacteriology 169(12): 5735(1987); and J. of Bacteriology 158:455 (1984)]. These 5 genes have been cloned and overproducing expression plasmids for these 5 subunits are commercially available. However, prior to the present invention, the identification for the remaining 5 subunits which make up the holoenzyme was not known.

The present invention describes, for the first time, the genetic and peptide sequences for the remaining five subunits of the polymerase III holoenzyme. In addition, to sequence these genes, very efficient operproducing plasmids for each of them have been constructed, and purification protocols for each have been devised. Whereas the low amount of holoenzyme in cells has allowed the subassemblies to be available in microgram quantities prior to the present invention (milligrams of pure α, ε, τ, δ and β subunits are available using molecular cloned genes in overproducing expression plasmids), utilizing techniques according to the present invention it has been possible to obtain approximately 100 mg of homogeneous subunit from 4 L of cells.

Prior to the identification of the remaining 5 genes of the holoenzyme, a few micrograms of each subunit was resolved from the holoenzyme. The sequence analysis of these resolved subunits eventually lead to the identification of their genetic sequences, and then to the genes per se. In addition, reconstitution studies were carried out to determine which subunits were essential to the speed and processivity of the holoenzyme. In addition, overproducing expression plasmids for these 5 subunits were produced.

Following these studies, it has now been determined, according to the present invention, that at least 5 subunits are required for the action of this enzyme (α, ε, β, δ, and γ), and preferably 6 subunits are essential for the speed and processivity of the holoenzyme. These subunits, the combination of which are essential for the unique synthetic capabilities of the holoenzyme, according to the present invention, are: α, ε, β, δ, δ', and γ.

The 5 subunits according to the present invention which have been identified, sequenced, cloned, provided in overproducing expression plasmids, expressed, and purified for the first time are subunits δ, δ', χ, θ, and ψ.

The following figures, detailed description and examples are provided in order to allow the reader to obtain a fuller and more complete understanding of the present invention. With regard to the figures,

FIG. 1 depicts the pET-δ expression vector according to the present invention;

FIG. 2 depicts the pET-δ' expression vector according to the present invention;

FIGS. 3A, B, and C depict the replication activity of δ, δ and δδ' with γ and τ according to the present invention;

FIGS. 4A and B depict the effect of δ' and δ on the ATPase activity of γ, and τ according to the present invention;

FIG. 5 depicts the pET-θ expression plasmid according to the present invention;

FIGS. 6A and B depict the reconstitution assay according to the present invention indicating that θ does not stimulate DNA synthesis;

FIG. 7 depicts that θ, according to the present invention stimulates ε excision of an incorrect 3' TG base pair;

FIGS. 8A and B depicts the native molecular weight of ae and polIII core according to the present invention;

FIG. 9 depicts the construction of the pET-ψ overproducing plasmid according to the present invention;

FIG. 10A and B depict the stimulation of the DNA dependent ATPase of γ and τ by ψ and χ, according to the present invention;

FIG. 11 depicts the construction of the pET-χ expression plasmid according to the present invention; and

FIGS. 12A and B depicts native molecular mass of χ, ψ and the χψ complex, according to the present invention.

More specifically with regard to FIG. 1, there is shown the expression vector for δ as prepared and described in the following examples. The holA gene excised from M13-δ-NdeI using NdeI is shown above the pET3c vector. The open reading frame encoding δ is inserted into the NdeI site of pET3c such that the initiating ATG is positioned downstream of the Shine-Dalgarno sequence and a T7 promoter. Downstream of the holA insert are 492 nucleotides of E. coli DNA and 591 nucleotides of M13mp18 DNA. The T7 RNA polymerase termination sequence is downstream of the holA insert.

More specifically with regard to FIG. 2, the holB fragment excised from M13-δ'-NdeI using NdeI is shown above the expression vector. The open reading frame encoding δ' is inserted into the NdeI site of pET3c such that the initiating ATG is positioned downstream of the Shine-Dalgarno sequence and a T7 promoter. The holB insert also contains 158 nucleotides of E. coli DNA downstream of the the holB stop codon to an NdeI site. The T7 polymerase termination sequence is downstream of the holB insert.

With regard to FIG. 3, replication assays were performed as described below. FIG. 3C summaries the replication assays. Either γ or τ was titrated into assays containing SSB "coated" primed M13mp18 ssDNA, ∫, ae and either 2 ng δ, 2 ng δ' or an equal mixture (1 ng each) of δ and δ' (δδ'). The reaction mixture was preincubated for 8 minutes to allow reconstitution of the processive polymerase prior to initiating a 20 second pulse of DNA synthesis. FIG. 3A depicts the results of the γ subunit being titrated into the replication mixture either alone (open squares) or containing either δ' (closed circles), δ (open circles), or δδ' (closed squares). FIG. 3B depicts the results of the γ subunit being titrated into the replication mixture either alone (open triangles), or containing either δ' (closed circles), δ (open circles), or δδ' (closed squares).

With regard to FIG. 4, ATPase assays were performed in the presence of M13mp18 ssDNA as described in detail below. The subunits in each assay are identified below the plot in the figure. FIG. 4A refers to the effect of δ, δ' and β on the γATPase; FIG. 4A refers to the effect of δ, δ' and β on the τATPase.

With regard to FIG. 5, the shaded NdeI-BamHI segment includes the holE gene (arrow). Transcription of the holE is driven by a T7 promoter. The T7 RNA polymerase termination sequence is downstream from the E. coli DNA insert. Translation of δ is aided by an upstream Shine-Dalgarno sequence.

With regard to FIG. 6, the replication reactions were performed as described below. FIG. 6A outlines the protocol summarizing the assay. Either the αε complex or reconstituted polIII core (αεθ) were titrated into the assay which contains β, γ complex and primed phage X 174 ssDNA "coated" with SSB. Proteins and DNA were preincubated for 6 minutes to allow time for assembly of the processive polymerase. A 15 second round of synthesis was initiated upon addition of remaining deoxynucleoside triphosphates. Circles: titration of αε complex; triangles: titration of αεθ. The α subunit was limiting in these assays and therefore the amount of αε and αεθ added to the assay is taken as the amount of α added.

With regard to FIG. 7, there is depicted the results of a titration of θ into the assay containing ε and a mismatched 3'32P-end-labelled T residue on a synthetic "hooked" primer template.

With regard to FIG. 8, the is shown a comparison of the migration of αε and polIII core relative to protein standards on gel filtration and in glycerol gradients. The position of polIII core reconstituted using either excess or substoichiometric θ was the same in both types of analysis. FIG. 8A depicts gel filtration analysis on Superose 12. The Stokes radius of protein standards was calculated from their known diffusion coefficients. FIG. 8B depicts glycerol gradient sedimentation analysis. Sedimentation coefficients of the standards are Amy, sweet potato β-amylase (152 kDa, 8.9S); Apf, horse apoferritin (467 kDa, 59.5 Å); IgG; bovine immunoglobulin G (158 kDa, 52.3 Å, 7.4S); BSA, bovine serum albumin (67 kDa, 34.9 Å, 4.41S); Ova, chicken ovalbumin (43.5 kDa, 27.5 Å, 3.6S); Myo, horse myoglobin (17.5 kDa, 19.0 Å, 2.0S). The positions of ae and polIII core relative to the protein standards are indicated in the plots. The Stokes radii and S values of αε and polIII were measured by comparison to the standards.

With regard to FIG. 9, the holD gene was amplified from genomic DNA using primers which form an NdeI site at the initiating ATG and downstream BamHI site. Due to an internal NdeI site within holD, insertion of the complete holD gene into the pET3c expression plasmid required the two steps shown below. Additional details appear in the following description.

With regard to FIG. 10, ATPase assays were performed using a two-fold molar excess of χ and ψ (as monomers) over γ and τ (as dimers) and using M13mp18 ssDNA as an effector. FIG. 10A depicts ATPase assays of ψ, χ, γ and combination of these proteins; FIG. 10B depicts the effect of ψ and χ subunits on the ATPase of τ. Subunits in the assays are indicated below the plots, and assays performed in the presence of SSB are indicated.

With regard to FIG. 11, the holC gene was amplified from genomic DNA using primers which generate an NdeI site at the start codon of holC and a BamHI site 152 nucleotides downstream of holC as described below. The 604 bp amplified product was purified, digested with NdeI and BamHI, and ligated into the NdeI and BamHI sites of pET3c to yield pET-χ. The open reading frame encoding χ was inserted into the NdeI-BamHI sites of pET3c such that the initiating ATG is positioned downstream of the Shine-Dalgarno sequence and a T7 promoter. The T7 RNA polymerase termination sequence is downstream of the holC insert. The Amp^(r) indicates the ampicillin resistance gene; the ori indicates the pB322 origin of replication.

With regard to FIG. 12A, the Stokes radius of χ, ψ and χψ complex was determined by comparison with protein standards in gel filtration on Superdex 75. With regard to FIG. 12B, the S value of χ, ψ and χψ complex determined by comparison to protein standards in glycerol gradient analysis are given. The protein standards were: bovine serum albumin (BSA), 34.9 Å, 4.41S; chicken ovalbumin (Ova) 27.5 Å, 3.6S; soybean trypsin inhibitor (Ti), 23.8 Å; bovine carbonic anhydrase II (Carb), 3.06S; horse myobglobin (Myo), 19.0 Å, 2.0S; and horse kidney metalothionin (Met), 1.75S.

In general, the sequence for each of the genes for the five subunit peptides, according to the present invention, began with isolating, purifying and sequencing the individual peptides.

The δ, δ', χ, ψ subunits were purified by a combination of two published procedures. First the γ complex (γ, δ, δ', ψ), was purified from 1.5 Kg E. coli HB101 (pNT203-pSK100) as described by Maki [see J. Bio. (Chem 263:6555(1988)]. Second, the complex was split into two fractions--a "γχψ" complex and a "δδ" complex--as described by O'Donnell [see J. Bio. (Chem 265:1179 (1990)]. The peptide sequences for δ and δ' were obtained from the δδ' fraction, whereas the peptide sequences of χ and ψ are obtained from the γψχ fraction. The θ subunit sequence was obtained from a side fraction off this procedure which contained nearly pure polymerase III (α, ε, θ) subunits.

For all 5 proteins, the amino acid sequences were obtained in the same manner, by the use of N-terminal analysis and tryptic analysis. N-terminal analysis was conducted using known techniques of SDS-PAG electrophoresis [see Nature 227:680(1970)] in a 15% gel, and subsequent electroelution onto PVDF membrane. The resolved peptides were removed from the membrane and sequenced. For tryptic analysis, either δδ' or γχψ comples was chromatographed in a 15% SDS-PAG gel to separate the individual subunits. However, for this procedure, the 100 pmol was electroblotted onto nitrocellulose. The nitrocellulose membrane was then digested with trypsin, and the peptides resolved by microbore HPLC. The resolved peptides were then sequenced.

The electroblotting procedure used in the tryptic analysis protocol is more fully described in the following general example:

EXAMPLE I

(electroblotting)

SDS (Bio-Rad) was purified by crystallization from ethanol-water. SDS (100 g) was added to ethanol (450 g), stirred, and heated to 55° C. Additional hot water was added (50-75 ml) until all of the SDS dissolved. Activated charcoal (10 g) was added to the solution, and after 10 minutes the slurry was filtered through a Buchner funnel (Whatman No. 5 paper) to remove all the charcoal. The filtered solution was chilled, first a 4° C. for 24 hrs and then at -20° C. for an additional 24 hrs. Crystalline SDS was collected on a coarse-frit sintered-glass funnel and washed with 800 ml of ethanol chilled to -20° C. The partial purified SDS was then recrystallized using the above procedure but without the charcoal. 0.75 mm SDS-Laemmli gel was made using ultra-pure reagents. Prior to electrophoresis 10 mM Glutatnhione (to a final concentration of 0.05 mM) was added to the upper chamber buffer, and the system preelectrophoresed 2 hr at 3-5 mA (3 mA for mini-gel, 5 mA for normal). After 2 hrs, the upper chamber was emptied and standard tris-glycine buffer was added. The samples to be run were denatured using Laemmli denaturaton solution made from the ultra-pure reagents (in the presence of 5 mM DTT). The gel was run under conditions such that separation was achieved in less than 2 hrs. After the gel run, the gel was soaked for 30 min in 10 mM CAPS pH 11, 5% methanol (% of methanol will vary depending on the size of the protein: in general, high molecular weight proteins transfer more efficiently in absence of methanol while low molecular weight proteins require methanol in the buffer). CAPS buffer was made by titrating a 10 mM solution with 10N NaOH. For gel transfer, slices of Immobilon were wet in 100% methanol and equilibrated 10 min in the CAPS transfer buffer, and the protein transferred using Bio-Rad mini blotter (transfer time will vary depending on protein size, methanol, etc.; -70 kDa polypeptide will transfer in 90 min in the presence of 5% methanol at 15 V). After transfer, Immobilon was soaked in distilled water for 5 min, and the membrane was stained with 0.1% Commassie Blue R250 in 50% methanol for 1 min, and destained in 50% methanol and 10% aldehyde-free acetic acid. The membranes were soaked in distilled water for 10 min, and allowed to air dry. Protein bands of interest were cut from the membrane and stored in eppendorf tubes at -20° C. until sequenced.

The identification of the subunit gene of DNA polymerase III δ was accomplished by purifying the δδ proteins to apparent homogeneity through an ATP-agarose column from 1.3 kg of the δ/τ overproducing strain of E. coli [HB 101 (pNT 203, pSK 100)].

The δδ' subunits were separated by electrophoresis in a 15% SDS-PAG (polyacrylamide gel), then electroblotted onto PVDF membrane (Whatman) for N-terminal sequencing (50 pmol each) [see J. Biol. (Chem. 262:10035 (1987)], and onto nitrocellulose membrane (Schleicher and Schuell) for tryptic analysis (140 pmol each) [see PNAs U.S.A. 84:6970 (1987)]. Proteins were visualized by Ponceau S (Sigma). Protein sequences were determined using an Applied Biosystems 470A gas-phase microsequenator. Sequence results were as follows: ##STR1##

The DNA sequencing, construction of the overproducing vector, and DNA replication assays for this subunit were conducted according to the following example:

EXAMPLE II

DNA sequencing:

The 3.2 kb KpnI/Bg1II (restriction enzymes, New England Biolabs) fragment containing δ was excised from γ169 (Kohara) and directionally ligated into pUC18 to yield pUCdelta. Both strands of DNA were sequenced by the chain termination method of Sanger using the United States Biochemicals sequenase kit, [α-³⁵ S]dCTP (New England Nuclear), and synthetic DNA 17-mers (Oligos etc. Inc.). All sequence information presented here was determined on both strands using both dGTP and dITP in sequencing reactions.

Construction of the overproducing vector:

Approximately 1.7 kb of DNA upstream of δ was excised from pUCdelta using KpnI (polylinker site) and BstXI (the BstXI site is 13 base pairs upstream of the start codon of holA) followed by self-ligation of the plasmid. A 1.5 kb fragment containing the holA gene was then excised using EcoRI and Xbal (these sites are in the pUC polylinker on either side of the δ insert) followed by directional ligation into M13mp18 to yield M13delta. An NdeI site was generated at the start codon of holA by primer directed mutagenesis [see Methods Enzymol 154:367 (1987)] using a DNA 33-mer (5'→3'): ##STR2## containing the NdeI site (underlined) at the start codon of holA to prime replication of M13delta viral ssDNA, and using DNA polymerase and SSB in place of Klenow polymerase to completely replicate the circle without strand displacement [see J. Biol. Chem. 260:12884 (1985)]. The NdeI site was verified by DNA sequencing. An NdeI fragment (2.1 kb) containing the S gene was excised from the NdeI mutated M13 delta and ligated into pET-3c linearized using NdeI to yield pETdelta. The orientation of the holA gene in pETdelta was determined by sequencing.

DNA replication assays:

The replication assay contained 72 ng M13mp18 ssDNA (0.03 pmol as circles) uniquely primed with a DNA 30-mer [see J. Biol. Chem. 266:11328 (1991)], 980 ng SSB (13.6 pmol as tetramer), 22 ng β (0.29 pmol as dimer), 200 ng γ(2.1 pmol as tetramer), 55 ng αε complex in a final volume (after addition of proteins) of 25 μl 20 mM Tris-HCL (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 4% glycerol, 40 μg/ml BSA, 0.5 mM ATP, 60 μM each dCTP, dGTP, dATP and 20 μM [α-³² P]dTTP (New England Nuclear). Proteins used in the reconstitution assay were purified to apparent homgeniety and their concentration determined. Delta protein or column fraction containing δ, was diluted in buffer (20 mM Tris-HCL (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol, 60 mM NaCl and 50 μg/ml BSA) such that 1-10 ng of protein was added to the assay on ice, shifted to 37° C. for 5 minutes, then quenched upon spotting directly onto DE81 filter paper. DNA synthesis was quantitated as described.

Gel filtration:

Gel filtration of δ, δ' and the δδ' complex was performed using an HR 10/30 Superdex 75 column equilibrated in 20 mM Tris-HCL (pH 7.5), 10% glycerol, 2 mM DTT, 0.5 mM EDTA and 100 mM (buffer B). Either δ (30 μg, 0.78 nmol as monomer), δ (30 μg, 0.81 nmol as monomer) or a mixture of δ and δ' (30 μg of each) were incubated for 30 minutes at 15° C. in 100 μl of buffer B then the entire 100 μl sample was injected onto the column. The column was developed with buffer B at a flow rate of 0.3 ml/minute and after the first 6 ml, fractions of 170 μl were collected. Fractions were analyzed by 13% SDS polyacrylamide gels (100 μl per lane) stained with Coomassie Brillant Blue. Densitometry of stained gels was performed using a Pharmacia-LKB Ultrascan XL laser densitometer.

Gel filtration of γ or γ mixed with either δ or δ' or both δ and δ' was performed using an HR 10/30 Superose 12 column equilabrated in buffer B. Protein mixtures were preincubated 30 minutes at 15° C. in 100 μl buffer B then injected onto the column and the column was developed and analyzed as described above. Replication activity assays of these column fractions were performed as described above with the following modifications. The γ subunit was omitted from the assay and each fraction was diluted 50-fold with 20 mM Tris-HCL (pH 7.5), 10% glycerol, 2 mM DTT, 0.5 mM EDTA and 50 μg/ml BSA. Then 2 μl of diluted fraction was withdrawn and added to the assay.

Protein standards were a mixture of proteins obtained from BioRad and from Sigma Chemical Co. and were brought to a concentration of approximately 50 μg each in 100 μl buffer B before analysis on either Superdex 75 or Superose 12 columns.

Glycerol gradient sedimentation:

Sedimentation analysis of δ, δ' and a mixture of δ and δ' were performed using 11.6 ml 10%-30% glycerol gradients in buffer B. Either δ (57 μg, 1.5 nmol as monomer), δ' (56 μg, 1.5 nmol as monomer) or a mixture of δ and δ' (57 μg and 56 μg, respectively) were incubated at 15° C. for 30 minutes in a final volume of 100 μl buffer B then each sample was layered onto a separate gradient. Protein standards (50 μg each in 100 μl buffer B) were layered onto another gradient and the gradients were centrifuged at 270,000×g for 60 hours at 4° C. Fractions of 170 μl were collected from the bottom of the tube and analyzed (100 μl/lane) in a 13% SDS polyacrylamide gel stained with Coomassie Blue.

Light scattering:

The diffusion coefficient of δ, δ' and the δδ' complex was determined by dynamic light scattering at 780 nm in a fixed angle (90°) Biotage model dp-801 light scattering instrument (Oros Instruments). Samples of δ (200 μg, 5.2 nmol as monomer), and δ (200 μg, 5.4 nmol as monomer) were in 400 μl of 20 mM Tris-HCL (pH 7.5), 100 mM NaCl and 1.2% glycerol. The mixture of δ and δ' (100 μg of each) was in 400 μl of 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The observed diffusion coefficient of δ' in the presence of 1.2% glycerol was 0.6% higher than in the absence of glycerol. Hence, the 1.2% glycerol in the δ and δ' samples had little effect on the observed diffusion coefficient.

The purification of δ was preformed according to the following example:

EXAMPLE III

(purification of δ)

BL21 (DE3) cells harboring pETdelta were grown at 37° C. in 12 liters of LB media containing 100 μg/ml of ampicillin. Upon growth to OD 1.5, the temperature was lowered to 25° C., and IPTG was added to 0.4 mM. After a further 3 hrs. of growth, the cells (50 g) were collected by centrifugation. Cells were lysed using lysozyme as described in prior publications, and the debris removed by centrifugation. The following purification steps were performed at 4° C. The assay for δ is as described above.

The clarified cell lysate (300 ml) was diluted 2-fold with 20 mM Tris-HCl (pH 7.5), 20% glycerol, 0.5 mM EDTA, 2 mM DTT (buffer A) to a conductivity equal to 112 mM NaCl, and then loaded (over 3 hrs.) onto a 60 ml Hexylamine Sepharose column equilibrated with buffer A plus 0,1M NaCl. The Hexylamine column was washed with 60 ml buffer A plus 0.1M NaCl, and then eluted (over 14 hrs) using a 600 ml linear gradient of 0.1M NaCl to 0.5M NaCl in buffer A. Eighty fractions were collected. Fractions 16-34 (125 mls) were dialyzed against 2 liters of buffer A plus 90 mM NaCl overnight, and then diluted 2-fold with buffer A to yield a conductivity equal to 65 mM NaCl just prior to loading (over 45 min) onto a 60 ml column of Heparin Sepharose equilibrated in buffer A plus 50 mM NaCl. The heparin column was washed with 120 ml buffer A plus 50 mM NaCl, and then eluted (over 14 hrs) using a 600 ml linear gradient of 0.05M NaCl to 0.5M NaCl in buffer A. Eighty fractions were collected. Fractions 24-34 were pooled and diluted 3-fold (final volume of 250 mls) with buffer A to a conductivity equal to 85 mM NaCl just prior to loading (over 50 min) onto a 50 ml Hi-Load 26/10 Q Sepharose fast flow FPLC column. The column was washed with 150 ml buffer A plus 50 mM NaCl, and then eluted using a 600 ml linear gradient of 0.05M NaCl to 0.5 mM NaCl in buffer A. Eighty fractions were collected. Fractions 28-36 which contained pure δ were pooled (74 mls, 1.9 mg/ml), passed over a 1 ml ATP-agarose column (N-6 linked) to remove any possible γ complex contaminant, and then dialyzed versus two changes of 2 liters each of buffer A containing 0.1M NaCl (the DTT was omitted for the purpose of determining protein concentration spectrophotometrically) before storing at -70° C.

The following table gives the results obtained from measuring the protein levels obtained from the fractions taken in Example III.

    ______________________________________                                                   total            specific                                                                              fold                                                   protein total    activity                                                                              purifica-                                    Fractions (mg)    units.sup.1                                                                             (units/mg)                                                                            tion   % yield                               ______________________________________                                         I   Lysate.sup.2                                                                             2070    5.4 × 10.sup.9                                                                   2.6 × 10.sup.6                                                                 1.0    100                                 II  Hexylamine                                                                               446     2.5 × 10.sup.9                                                                   5.6 × 10.sup.6                                                                 2.2    46                                  III Heparin   197     2.0 × 10.sup.9                                                                  10.2 × 10.sup.6                                                                 3.9    37                                  IV  Q Sepharose                                                                              141     1.5 × 10.sup.9                                                                  10.6 × 10.sup.6                                                                 4.1    28                                  ______________________________________                                          .sup.1 One unit is defined as pmol nucleotide incorporated per minute          .sup.2 Ommission of gamma from the assay of the lysate resulted in a           200fold reduction of specific activity (units/mg)                        

The δ gene was identified using amino acid sequence information from δ. The sequence of the N-terminal 28 amino acids of δ and the sequence of three internal tryptic peptides were determined. One of the tryptic peptides (tryptic peptide δ-1) overlapped 10 amino acids of the N-terminal sequence. A search of the GenBank revealed a sequence which predicted the exact amino acid sequence of the 21 amino acid tryptic peptide δ-1 which overlapped the N-terminal sequence. The matching sequence occurred just downstream of the rlpB gene at 15.2 minutes of the E. coli chromosome. The match of the published DNA sequence to the N-terminal sequence of δ was imperfect due to a few errors in the published sequence of this region. The published sequence of r1pB accounted for approximately 22% of the δ gene and did not encode either of the other two tryptic fragments. The Kohara lamda phage 169 contains 19 kb of DNA surrounding the δ gene. The 3.2 kb KpnI/Bg1II fragment containing δ was excised from λ169, cloned into pUC18 and the δ gene was sequenced. The DNA sequence predicts the correct N-terminal sequence of δ (except the Ile instead of Leu at position 2) and encodes the other two internal tryptic peptides of δ in the same reading frame, and predicts a 343 amino acid protein of 38.7 kda consistent with the mobility of the δ in SDS-PAGE (35 kDa).

The full nucleic acid sequence for the δ gene according to the present invention was determined to be: ##STR3##

The underlined portions of this sequence refer to subunits which are δ-1 (55-117), δ-2 (358-399), and δ-3 (604-672). In addition, the upstream sequence: ##STR4## wherein the last underlined TG denotes two-thirds of rlpB stop codon; in addition, the positive RNA polymerase promoter signals (TCGCCA and GATATT) and the Shine-Dalgarno sequence (ACGCT) are underlined.

In addition, the downstream nucleic acid sequence for holA is as follows: ##STR5##

The holA gene translates into the amino acid sequence: ##STR6##

The holA gene is located in an area of the chromosome containing several membrane protein genes. They are all transcribed in the same direction. The mrdA and mrdB genes encode proteins responsible for the rod shape of E. coli and the r1pA and r1pB genes encode rare lipoproteins which are speculated to be important to cell duplication. The position of the δ gene within a cluster of membrane proteins may be coincidental or may be related to the putative attachment of the replisome to the membrane.

As noted, the termination codon of the r1pB protein overlaps one nucleotide with the initiating ATG of holA leaving a gap of only 2 nucleotides between these genes. holA may be an operon with r1pB or there may be a promoter within r1pB. The nearest possible initiation signals for transcription (the putative RNA polymerase signals) and translation (Shine-Dalgarno) are underlined in the sequence given above; the match to their respective consensus sequences is not strong suggesting a low utilization efficiency. Inefficient transcription and/or translation may be expected for a gene encoding a subunit of a holoenzyme present at only 10-20 copies/cell. The δ gene uses several rare codons [CCC(Pro), ACA(Thr), GGA(Gly), AGT(Ser), AAT(Asn), TTA, TTG, CTC(all Leu)] 2-5 times more frequently than average which may decrease translation efficiency. ATP binding to δ within the holenzyme has been detected previously by UV crosslinking. The DNA sequence of the δ shows a near match to the ATP binding site consensus motif (i.e. AX₃ GKS for δ at residues 219-225 compared to the published consensus G/AX₄ GKS/T, G/AXGKS/T or G/AX₂ GXGKS/T [see Nuc. Acids Res 17:8413 (1989)]. Whether δ binds with ATP specifically at this site remains to be determined. Of the 33 arginine and lysine residues in δ, 16 of them (50%) are within amino acids 225-307. This same region contains only 5 (14%) of the 35 glutamic and aspartic acid residues. Whether this concentration of basic residues is significant to function is unknown. There are no strong matches to consensus sequences to motifs encoding: zinc fingers or helix-loop-helix DNA binding domains.

The holA gene was cloned into M13mp18 and an NdeI site was created at the initiating methionine by the site directed mutagenesis technique in order to study the overproduction of this gene. The δ gene was then excised from M13delta and inserted into the NdeI site of the pET-3c expression vector [see Methods Enzymol 185:60 (1990)] which places δ under control of a strong T7 RNA polymerase promotor, see FIG. 3-1. Upon transformation into BL21(DE3) cells and induction of T7 RNA polymerase with IPTG, the δ protein was expressed to 27% total cell protein. For reasons unknown, δ was not produced in BL21(DE3) cells containing the pLysS plasmid. Induction at 25° C. yielded approximately 2-fold more δ and increased the solubility of the overproduced δ relative to induction at 37° C. Twelve liters of induced cells were lysed using lysozyme and 141 mg of pure δ was obtained in 28% overall yield upon column fractionation using Hexylamine Sepharose, Heparin Agarose, and Q Sepharose. Delta protein tended to precipitate upon standing in low salt (<70 mM), especially during dialysis. Therefore, low salt was avoided except for short periods of time and column fractions containing δ were sometimes diluted in preparation for the next column rather than dialyzed overnight. The δ subunit was assayed by its ability to reconstitute efficient replication of a singly primed M13mp18 ssDNA "coated" with SSB in the presence of α, ε, β, and γ subunits. Cell lysate prepared from induced cells containing pETdelta were more active in the replication assay than cell lysate prepared from induced cells containing the pET-3c vector.

The expressed δ protein comigrated with the authentic δ subunit contained within the γ complex of the holoenzyme. The N-terminal sequence analysis of the pure cloned δ was identical to that predicted from the holA sequence according to the present invention provided that the protein encoded by the gene had been purified. Furthermore, the overproduced δ subunit was active with only the α, ε, γ and β subunits of the holenzyme (FIG. 5-1). In the presence of a sixth subunit, δ', activity was enhanced. The amount of the cloned δ required to reconstitute the efficient DNA synthesis characteristic of the holoenzyme using the 5 or 6 subunit combination according to the present invention is in the range shown previously for the naturally purified δ resolved from the γ complex. As shown below, addition of more γ to the replication assay brings the amount of δ down even further to about 1 ng for a stoichiometry of about 1-2 δ monomers per DNA circle replicated.

Electrospray mass spectrometry of the cloned δ protein yielded a molecular mass of 38,704 da. This mass is within 0.0015 of the mass predicted from the gene; well within the 0.01% error of the mass spectrometry technique. This is evidence that the DNA sequence above according to the present invention contains no errors and indicates the overproduced δ is not modified during or after translation. The ε₂₈₀ calculated from the amino acid composition of δ is 46,230 M⁻¹ cm⁻¹. The measured absorbance of δ in 6M guanidine hydrochloride is only 0.2% higher than in buffer A. Hence, the ε₂₈₀ of native δ is 46,137 M⁻¹ cm⁻¹.

Further understanding of the individual subunits the present invention also determines whether δ and δ' are monomeric, dimeric or higher order structures. The δ and δ' subunits were also each analyzed in a gel filtration column, and they migrated in essentially the same position as one another (fractions 30-32). As discussed below the δ' appears as two proteins, δ'_(L) and δ'_(S), which differ by approximately 0.5 kda. Comparison with protein standards of known Stokes radius yielded a Stokes radius of 26.5 Å, for δ and 25.8 Å, for δ, slightly smaller than the 27.5 Å radius of the 43.5 kDa ovalbumin standard indicating both δ and δ' are both monomeric (their gene sequences predict: δ, 38.7 kDa δ', 36.9 kDa). In a glycerol gradient sedimentation analysis both δ and δ' migrated in the same position as one another with an S value of 3.0 relative to protein standards, a slightly lower sedimentation value than the 43.5 kda ovalbumin standard, again indicating a monomeric state for the δ and δ'. Besides protein mass, the protein shape is also a determinant of both the Stokes radius and the S value obtained by these techniques. The shape however, causes opposite behavior in these two techniques, a protein with an asymmetric shape behaves in gel filtration as a larger protein than if it were spherical (elutes early) and behaves in sedimentation like a smaller protein than if it were spherical (sediments slower). The Stokes radius and S value can be combined in the equation of Siegel and Monty whereupon the protein shape factor cancels. Therefore, the native mass of the protein obtained from such treatment is more accurate than calculating the mass from only the S value or the Stokes radius and assuming a spherical shape. This calculation yielded a native mass of 34.7 kDa for δ and 33.8 kDa for δ'; values similar to the monomer molecular mass predicted from the gene sequences of δ and δ', further evidence they are monomers. Their frictonal coefficients are each significantly greater than 1.0 indicating they are not spherical but have some asymmetry to their shape. One can also conclude from this work that the two δ' subunits are a mixture of δ'_(L) and δ'_(S) rather than a complex of δ'_(L) and δ'_(S).

In initial studies using the cloned δ, δ' forms only a weak complex with γ but, together with δ' a stable γδδ' complex can be reconstituted which remains intact in gel filtration and ion exchange chromatography. Likewise, δ' forms only a weak complex with γ, and requires the δ subunit to bind γ tightly. Both δ and δ' appear monomeric and bind to each other to form a δδ' heterodimer.

Availability of the δ subunit in large quantity will allow detailed studies of the mechanism of the γ complex in β clamp formation. Further, identification of the α gene will provide for genetic analysis (essentiality) of δ in E. coli replication and possibly other roles of δ in DNA metabolism.

The second subunit according to the present invention, that of δ', was also identified from the δδ' fraction in like manner. The N-terminal sequence, comprising the first 18 amino acids in the peptide, and the tryptic peptide sequence were obtained. The amino acid sequence determined from the initial sequence studies for the δ' peptide is: ##STR7##

From these sequences, two DNA oligonucleotide probes were made and used (after end-labelling with ³² P for use in Southern blot analysis) to probe a Southern blot of E. coli DNA which was grown, isolated and restricted as above. The sequences of the two probes were: ##STR8##

Of the two Southern blots analyzed (one with the 57-mer probe and the other with the 54-mer probe), the patterns from the blots had one set of bands in common, and these were sized by comparison with size standards in the same gel following recognized techniques. The size of these 8 common "bands" or DNA fragments produced by digestion with 8 restriction enzymes were used to scan, by eye, the restriction map of the E. coli genome [see Cell 50:495 (1987)]. One unique location on the genome was located which was compatable with all 8 restriction fragment sizes.

Phage λ₂₃₆ was selected as a phage containing the "unique location" in the E. coli genome. The δ' gene was excised from the λ₂₃₆ phage using restriction enzymes EcoRV and KpnI to yield a 2.3 kb fragment of DNA. This fragment was then ligated into pUC18 and sequenced using a sequenase kit (US Biochemicals) in accordance with the manufacturer's instructions. The fragment was also ligated into a M13mp18 vector for making a site specific mutation, as described above, at the ATG start codon (i.e., changing the CGCATG to CATATG; thereby allowing NdeI to cleave the nucleotide at CATATG, whereas it could not cleave the nucleotide using the normal CGCATG sequence).

The nucleic acid sequence obtained from these studies predicted the amino acid sequence determined for δ' peptide in frame, and thus the selected sequence was that for the δ' gene. The nucleic acid sequence, according to the present invention, for this second subunit, δ', is: ##STR9##

The underlined portions of this sequence refer to subunits which are δ'-1 (283-315), δ'-2 (316-327), δ'-3 (328-390), δ'-4 (391-462), δ'-γ (481-534), and δ'-6 (577-639). In addition, the upstream sequence: ##STR10## contains an underlined putative translational signal: Shine-Dalgarno.

In addition, the downstream nucleic acid sequence for δ' begins with a stop codon: ##STR11##

The δ' gene (holB) was then subcloned into M13mp18, and a NdeI site was created at the initiating codon as described above. The δ' gene was then excised from M13 using NdeI restriction enzyme and a second enzyme which cut downstream of δ', and the excised gene was subcloned into the pET-3c overexpression plasmid using the same techniques described above. Following overexpression of the δ' protein, the protein was purified using a Fast flow Q--Heparin--Hexylamine techique as described herein. Ninety mg of δ' protein was obtained from 4 liters of cells.

Further studies on the δ' gene were conducted to make certain that the gene sequence obtained from these research was actually the δ' gene and not some artifact. These studies showed that the gene sequence according to the present invention predicted all the peptide sequence information, that the cloned δ' gene comigrates with the naturally occuring gene on a 13% SDS-PAG gel, that the cloned δ' gene stimulates the 5 protein system as does the naturally occuring δ', and that δ' forms a δ'δ complex with δ in a similar manner to that which occurs with the naturally occurring δ' and δ.

With specific regard to the isolation and characterization of δ' and holB according to the present invention, the amino acid sequencing was conducted using δ and δ' subunits purified to apparent homogenicity through the ATP-agarose column step [see J. Biol. Chem. 265:1179 (1990)] from 1.3 kg of the γ/τ overproducing strain of E. coli: HB101(pNT203, pSK100), [see J. Biol. Chem. 263:6555 (1988)]. The δ and δ' subunits were separated on a 13% SDS polyacrylamide gel whereupon the δ' resolved into two bands. The slower and faster migrating δ' bands are referred to as δ'_(L) (large) and δ'_(S) (small), respectively; δ'_(S) was approximately 2 times the abundance of δ'_(L). Both δ'_(L) and δ'_(S) were electroblotted onto PVDF membrane (Whatman) [see J. Biol. Chem. 262:10035 (1987)] for N-terminal sequencing (50 pmol each of δ'_(L) and δ'_(S)), and onto nitrocellulose membrane (Schleicher and Schuell) [see Proc. Natl. Acad. Sci. U.S.A. 84:6970 (1987)] for tryptic analysis (90 pmol of δ'_(L) and 180 pmol of δ'_(S)). Proteins were visualized by Ponceau S stain (Sigma).

Analysis of the more abundant δ'_(S) was as follows: the N-terminal sequence was: ##STR12## and the tryptic peptides were: ##STR13##

Parenthesis in the above sequences indicate uncertain assignments.

Two synthetic oligonucleotide probes (DNA oligonucleotides, Oligos etc. Inc.) were designed from the sequence of two of the tryptic peptides and the codon usage of E. coli with allowance for a T-G mispair at the wobble position. A synthetic DNA 57-mer probe was based on the sequence of δ'-4 (amino acids 131-149): ##STR14## (after identification and sequencing of holB this probe was incorrect at 11 positions). A DNA 54-mer probe was based on the sequence of δ'-6 (amino acids 195-212): ##STR15## (after identification and sequencing of holB the probe was incorrect at 9 positions. These probes (100 pmol each) were 5' end-labelled with 1 μM [γ³² P]ATP (radionucleotides, Dupont-New England Nuclear) and polynucleotide kinase. E. coli genomic DNA (strain C600) was extracted [see J. Mol. Bio. 3:208 (1961)] and restricted with either BamHI, HindIII, EcoRI, EcoRV, BglI, Kpni, PstI or PvuII (DNA modification enzymes, New England Biolabs) and then each digest was electrophoresed in a 0.8% native agarose gel followed by depurination (0.25M HCl), denaturation (0.5M NaCl) and then neutralized (1M Tris, 2M NaCl, pH % 0.0) prior to transfer to Gene Screen Plus (DuPont-New England Nuclear) for Southern analysis using a Vacugene appartus (Pharmacia) in the presence of 2×SSC (0.3M NaCl. 0.3M sodium acetate, pH 7.0). Conditions for hybridization and washing using these oligonucleotide probes were determined empirically and the desired results were obtained using a hybridization temperature of 42° C. then washing with 2×SSC and 0.2% SDS at successively higher temperature until evaluation by autoradiography showed a single band in each lane for the 57-mer, and two bands in each lane for the 54-mer (this occurred at 53° C. for both probes). Although the 54-mer showed two bands in each lane, one band always matched the position of the band probed with the 57-mer.

The 2.1 kb KpnI/EcoRV fragment containing holB was excised from λ E9G1(236) [see Cell 50:495(1987)] and directionally ligated into PUC18 (KpnI/HincII) to yield pUC-δ'. Both strands of DNA were sequenced by the chain termination method of Sanger using the United States Biochemicals sequenase kit, [α⁻³⁵ S]dATP, and synthetic DNA 18-mers.

A 2.1 kb KpnI/HindIII fragment containing the holB gene was excised from pUC-δ' and directionally ligated into M13mp18 to yield M13-δ'. An NdeI site was generated at the start codon of holB by oligonucleotide site directed mutagenesis [see Methods Enzymol 154:367 (1987)] using a DNA 33-mer: ##STR16## containing the NdeI site (underlined) at the start codon of holB to prime replication of M13-δ' viral ssDNA and using SSB and DNA polymerase III holoenzyme (in place of DNA polymerase I) to replicate the circular template without strand displacement. The M13 chimera is called M13-δ'-NdeI. And NdeI fragment (1160 bp) containing the holB gene was excised from M13-δ' -NdeI and ligated into pET3c, linearized using NdeI, to yield pET-δ'. The orientation of the holB gene in pET-δ' was determined by sequencing.

Reconstitution assays contained 108 ng M13mp18 ssDNA (0.05 pmol as circles) uniquely primed with a DNA 30-mer [see J. Biol. Chem 266:11328 (1991)], 1.5 μg SSB (21 pmol as tetramer), 30 ng β (0.39 pmol as dimer), 22.5 ng αε complex (0.14 pmol), 20 ng γ (0.12 pmol as dimer), 2 ng δ (0.5 pmol as monomer) and the indicated amount of δ' (or 1-5 ng of column fraction during purification) in 20 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 4% glycerol, 40 μg/ml BSA, 0.5 mM ATP, 60 μM dGTP, and 0.1 mM EDTA in a final volume of 25 μl (after the addition of the remaining proteins). Assays of γ or τ activity with either contained either 2 ng δ (0.05 pmol as monomer), 2 ng δ' (0.05 pmol as monomer), or 1 ng (0.025 pmol) each of δ and δ', and the indicated amount of γ or τ. All proteins were added to the assay on ice and then shifted to 37° C. for 8 minutes to allow reconstitution of the pocessive polymerase on the primed ssDNA. DNA synthesis was initiated upon rapid addition of 60 μM dATP and 20 μM [α³² P]TTP, then quenced after 20 seconds and quantitated using DE81 paper. When needed, proteins were diluted in 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol, and 50 μg/ml BSA. Proteins used in the reconstitution assays were purified [see J. Biol. Chem 266:9833 (1991). The concentration of β and δ were determined by absorbance using an ε₂₈₀ value if 17,900 M⁻¹ cm⁻¹, and 46,137 M⁻¹ cm⁻¹, respectively. Concentrations of α, ε, γ, τ and SSB were then determined [see Anal. Biochem 72:248 (1976)] using BSA as a standard. The concentration of δ' was determined by absorbance using an ε₂₈₀ value of 60,136 M⁻¹ cm⁻¹.

ATPase assays were performed in a final volume of 20 μl containing 20 mM Tris-HCl (pH 7.5), 8 mM MgCl₂ and contained 285 ng M13mp18 ssDNA. ATPase assays of γ, δ, δ'δδ' , γδ and γδ' with and without β contained 100 μM [γ⁻³² P] ATP and when present 376 ng γ (4 pmol as dimer), 304 ng δ (7 pmol as monomer). 296 ng δ' (8.0 pmol as monomer), and 320 ng β (4.2 pmol as dimer). Proteins were added on ice, shifted to 37° C. for 30 minutes, then 0.5 ml was spotted on a plastic backed thin layer of chromatography (TLC) sheet coated with Cel-300 polyethyleneimine (Brinkman Instruments Co.). To assay the more active ATPase activity of γδδ' and τ, 300 μM ATP was used, less total protein and less time at 37° C. inorder to assess the initial rate of reaction. Therefore, ATPase assays of γδδ, τ, τδ, τδ' and τδδ' with and without β contained 300 mM [γ⁻³² P] ATP and when present, 47 ng γ (0.5 pmol as dimer), 71 ng τ (0.5 pmol as dimer), 38 ng δ (1 pmol as monomer), 37 ng δ' (1 pmol as monomer) and 40 ng β (0.5 pmol as dimer. Proteins were added on ice, shifted to 37° C. for 10 minutes, then analyzed by TLC as described above.

TLC sheets were developed in 0.5M lithium chloride, 1M formic acid. An autoradiogram of the TLC chromatogram was used to visualize the free phosphate at the solvent front and ATP at the origin which were then cut from the TLC sheet and quantitated by liquid scintillation. The amount of ATP hydrolyzed was calculated as the percent of total radioactivity located at the solvent front (P_(i)) times the total moles of ATP added to the reaction.

The results of the δ' studies appear below:

The naturally purified δ' (resolved from the γ complex) appears in a 13% SDS polyacrylamide gel as two bands of approximately 37 kDa that differ in size by about 1 kDa. The larger protein (δ'_(L)) is approximately one half the abundance of the smaller one (δ'_(S)). Both δ'_(L) and δ'_(S) are believed encoded by the same gene as there was no noticeable difference in their HPLC profiles upon digestion with trypsin. In support of this, peptides from δ'_(S) and δ'_(L) that had the same retention time on HLPC analysis also had identical amino acid sequences (peptide δ'-7 from δ'_(S) and δ'-3 from δ'_(L) were identical). The N-terminus of δ'_(S) and five tryptic peptides of δ'_(S) and two tryptic peptides of δ'_(L) were sequenced.

A search of the GenBank revealed no match to the N-terminal sequence or to any of the tryptic peptides from either δ'_(L) or δ'_(S). Two best-guess oligonucleotide probes (a 57-mer and a 54-mer) were designed from tryptic peptides δ'-4 and δ'-6 based on the codon usage frequency in E. coli [see PNAS U.S.A. 80:687 (1983)]. The oligonucleotide probes were used in a Southern analysis of E. coli genomic DNA digested with each of the eight Kohara restriction map enzymes. Imposing the restraint that the eight restriction fragments from the Southern analysis must overlap the holB gene, the Kohara map of the E. coli chromosome was searched and only one position of overlap at 24.3 minutes (1,174 kb on the E. coli chromosome starting from thrA) was found which satisfied the fragment sizes. The fragment sizes in the Kohara map and from the Southern analysis are given in the following table which depicts the correspondence of the observed size of genomic DNA restriction fragments with the Kohara restriction map of the E. coli chromosome in the region of 24 minutes. E. coli genomic DNA was digested with the restriction enzymes indicated. The size of the restriction fragments that were in common for both the 57-mer and 54-mer probes in the Southern analysis and also the corresponding sizes of the restriction fragments on the Kohara restriction map of the E. coli chromosome at 24.5 minutes are listed below.

    ______________________________________                                         Restriction Size of restriction fragment (kb)                                  enzyme        Southern Kohara map                                              ______________________________________                                         Pstl          1.7      1.9                                                     Bgll          4.25     4.2                                                     Kpnl          6.6      6.4                                                     EcoRV         7.0      6.8                                                     Pvull         6.2      6.2                                                     EcoRl         >15      16.2                                                    Hindlll       >20      30                                                      BamHl         >25      38                                                      ______________________________________                                    

The Kohara λ phage E9G1(236) contains 16.2 kb of DNA surrounding the putative holB gene. A 2.1 KpnI/EcoRV fragment containing holB was excised from λ E9G1(236), cloned into pUC18 and sequenced. The sequence of the KpnI/EcoRV fragment revealed an open reading frame of 1002 nucleotides which predicts a 334 amino acid protein of 36.9 kDa (predicted pl of 7.04), consistent with the mobility of δ' in a SDS polyacrylamide gel. The open reading frame encodes the N-terminal sequence and all six tryptic peptide sequences obtained from δ'_(L) and δ'_(S).

Analysis of the DNA sequence upstream of the open reading frame revealed a putative translation initiation signal (Shine-Dalgarno sequence) 8 nucleotides upstream of the ATG initiating codon. No obvious transcription initiation signals were detected upstream of the initiation codon leaving open the possibility that holB is in an operon with an upstream gene(s). Alternatively, the transcription initiation signals may poorly match the consensus signals and thereby be unrecognizable, as a low level of transcription would not be unexpected for a gene encoding a subunit of the holoenzyme present at only 10-20 copies/cell. The holB gene uses several rare codons [TTA (Leu), ACA (Thr), GGA (Gly), AGC, TCG (Ser)]2-4 times more frequently than average which may decrease translation efficiency.

The holB sequence contains a helix-turn-helix consensus motif (Ala/GlyX₃ GlyX₅ Ile/Val) at Ala₈₀ Gly₈₄ Val₉₀ although ability of δ' to bind DNA has yet to be examined. There is also a possible leucine zipper (Leu₇ X₆ Leu₁₄ X₆ Gly₂₁ X₆ Leu₂₈) in the N-terminus although Gly interrupts the Leu pattern. The holB sequence does not contain consensus sequences for motifs encoding an ATP-binding site or a zinc finger. The molar extinction coefficient of δ' calculated from its 8 Trp and 11 Tyr residues is 59,600 M⁻¹ cm⁻¹ which is only 0.9% lower than that observed in the presence of 6M guanidine hydrochloride for a native extinction coefficient of 60,136 M¹ cm⁻¹.

To obtain the δ' subunit in large quantity, an expression plasmid was constructed. The holB gene was first cloned into M13mp18 followed by site directed mutagenesis to create an NdeI site at the initiating methionine to allow precise subcloning of holB into the pET3c expression vector. The holB gene was excised from the M13-δ'-NdeI mutant using NdeI followed by insertion into the NdeI site of the pET3c expression vector [see Methods Enzymol 185:60 (1990)] which places holB under the control of the T7 RNA polymerase promotor of T7 gene 10 and the efficient Shine-Dalgarno sequence of gene 10. The pET-δ' construct was transformed into BL21(DE3)plysS cells which harbor a λ lysogen containing the T7 RNA polymerase gene controlled by the lac UV5 promoter. Upon induction of T7 RNA polymerase with IPTG, the δ' protein was expressed to 50% of total cell protein. Cell lysate prepared from the induced cells containing pET-δ' was 5600-fold more active in the replication assays than cell lysate prepared from induced cells containing the pET3c vector as described below.

Three hundred liters of BL21(DE3)plysS cells harboring pET-δ' were grown at 37° C. in LB media supplemented with 5 mg/ml glucose, 10 μg/ml thiamine, 50 μg/ml thymine containing 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. Upon growth to an OD₆₀₀ of 0.6, IPTG was added to 0.2 mM. After further growth for two hours the cells (940 g) were collected by centrifugation, resuspended in an equal weight of 50 mM Tris-HCl (pH 7.5), 10% sucrose (Tris-Sucrose) and stored at -70° C. 100 g of cells (30 liters of cell culture) were thawed whereupon they lysed (due to lysozyme produced by plysS) and to this was added 250 ml Tris-Sucrose, DTT to 2 mM and 40 ml of 10×heat lysis buffer (50 mM Tris-HCl (pH 7.5), 10% sucrose, 0.3M spermidine, 1M NaCl). The cell debris was removed by centrifugation to yield the cell lysate (Fraction I, 4.41 g in 325 ml). The purification steps that followed were performed at 4° C. The reconstitution activity assay for δ' is as described previously. Ammonium sulphate (0.21 g/ml) was dissolved in the clarified cell lysate and stirred for 90 minutes. The precipitated protein containing δ' was pelleted (Fraction II, 1.58 g) and redissolved in 660 ml of 30 mM Hepes-NaOH (pH 7.2), 10% glycerol, 0.5 mM EDTA, 2 mM DTT (buffer A) and dialyzed against two successive changes of 2 liters each of buffer A to a conductivity equal to 40 mM NaCl. The Fraction II was loaded onto a 300 ml heparin agarose column (BioRad) equilibrated with buffer A. The heparin column was washed with 450 ml buffer A plus 20 mM NaCl, then eluted over a period of 14 hours using a 2.5 liter linear gradient of 20 mM NaCl to 300 mM NaCl in buffer A. One hundred fractions were collected. Fractions 36-53 were pooled (Fraction III, 550 ml, 990 mg) and dialyzed twice against 2 liters of 20 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, 2 mM DTT (buffer B) to a conductivity equal to 60 mM NaCl. The Fraction III was loaded onto a 100 ml Q sepharose column (Pharmacia) equilabrated with buffer B. The loaded Q sepharose column was washed with 150 ml of buffer B plus 20 mM NaCl then eluted over a period of 12 hours using a 1.2 liter linear gradient of 20 mM NaCl to 300 mM NaCl in buffer B. Eighty fractions were collected. Fractions 34-56 were pooled (Fraction IV, 781 mg in 370 ml) and dialyzed twice against 2 liters each of buffer B to a conductivity equal to 60 mM NaCl just prior to loading onto a 60 ml EAH sepharose column (Pharmacia) that was equilibrated with buffer B B. The loaded EAH sepharose column was washed with 60 ml of buffer B plus 40 mM NaCl then eluted over a period of 10 hours using a 720 ml linear gradient of 40 mM NaCl to 500 mM NaCl in buffer B. Eighty fractions were collected. Fractions 18-30 (Fraction V, 732 mg in 130 ml), which contained homogeneous δ' were pooled and dialyzed against 2 L buffer B (lacking DTT to allow an absorbance measurement, see below) to conductivity of 40 mM NaCl. Fraction V was passed over a 5 ml ATP-agarose column (Pharmacia, Type II, N-6 linked) to remove any γ complex contaminant followed by addition of DTT to 2 mM and then was aliquoted and stored at -70° C. Protein concentration was determined using BSA as a standard except at the last step in which concentration was determined by absorbance using ε₂₈₀ =60,136 M⁻¹ cm⁻¹.

    ______________________________________                                                                           fold                                                           total           purifica-                                              total   units.sup.1                                                                             specific                                                                              tion                                         Step      protein (mg)     activity                                                                              (units/mg)                                                                            % yield                               ______________________________________                                         I   Lysate.sup.2,3                                                                           4414    3.0 × 10.sup.1                                                                   7 × 10.sup.6                                                                   1.0    100                                 II  Ammonium  1584    2.5 × 10.sup.10                                                                 16 × 10.sup.6                                                                   2.3    83                                      Sulfate                                                                    III Heparin   990     2.6 × 10.sup.10                                                                 26 × 10.sup.6                                                                   3.7    87                                  IV  Q Sepharose                                                                              781     2.6 × 10.sup.10                                                                 33 × 10.sup.6                                                                   4.7    87                                  V   EAH-      732     2.5 × 10.sup.10                                                                 34 × 10.sup.6                                                                   4.9    83                                      Sepharose.sup.4                                                            ______________________________________                                          .sup.1 One unit is defined as pmol nucleotide incorporated in 20 seconds       .sup.2 Lysate of BL21(DE3)plysS cells harboring the pET3c vector yielded       specific activity of 1252 units/mg.                                            .sup.3 Omission of γ and δ from the assay of the lysate            resulted in a 7650fold reduction of specific activity (915 units/mg).          .sup.4 Using pure δ', omission of γ from the assay gave no         detectable synthesis under the conditions of the assay.                  

The purified overproduced δ' stimulated γδ 30-fold in its action in reconstituting the processive holoenzyme from the αε polymerase and the β clamp accessory protein. In this assay the δ' is titrated into a reaction containing a low concentration of γ and δ and also contains the β subunit, αε polymerase and M13mp18 ssDNA primed with a synthetic olignucleotide and coated with SSB. The proteins were preincubated with the DNA for 8 minutes to allow time for the accessory proteins to form the preinitiation complex which contains the β clamp and for αε to bind the preinitiation complex. DNA synthesis is initiated upon addition of deoxyribonucleoside triphosphates and the reaction is stopped after 20 seconds which is sufficient time for the processive reconstituted polymerase to complete the circular DNA. Although a processive polymerase can be reconstituted without the δ' subunit, under the conditions used in the present invention in which γ and δ are at low concentration, the δ' subunit stimulates the reaction greatly (30-fold). The δ' subunit saturated this assay at a level of approximately one molecule of δ' to one molecule of δ.

Both the τ and γ subunits of the holoenzyme are encoded by the same gene (dnaX). The γ subunit is formed as a result of a -1 frameshift during translation with the result that γ is only 2/3 the length of τ due to an earlier translational stop codon (within 2 codons) in the -1 reading frame. The activity of the γ and τ proteins in reconstituting the processive polymerase was compared using either the δ, δ' or both δδ' subunits in the presence of αε complex and β subunit (FIGS. 6A and 6B). In the absence of δ and δ', the γ subunit alone displays insignificant activity in the reconstitution assay although when a large amount of γ was present it had very little, but detectable, activity (FIG. 6A). The δ subunit provides γ with activity in the reconstitution assay, but δ' does not provide γ with activity. However, the cloned δ' subunit, when present with δ, markedly stimulated the activity of the γ and δ mixture such that maximal activity was achieved at much lower concentrations of added γ.

The τ subunit alone, like γ, was also essentially inactive in the reconstitution assay, although at very high amounts of τ a slight, but reproducible amount of activity was observed. τ is active with δ in this assay although more τ (50-fold) than γ is needed for comparable activity. Previously it was observed that τ was unlike γ in that τ was active with δ' in the reconstitution assay in the absence of any δ subunit (only τ, δ' and α, ε, β were needed). Consistent with these previous results, the δ' subunit is active with τ in the absence of δ (similar to the activity of τ and δ in the absence of δ'). With both δ and δ' present, only a small amount of τ subunit is required for maximal activity in the reconstitution assay. The activity of τδδ' parallels that of γδδ' and requires 500-fold less τ for maximal activity than either τδ or τδ'. Hence, both the γ subunit and the τ subunit are highly active in this reconstitution assay when both δ and δ' are present.

The effect of the δ, δ' and β subunits on the DNA dependent ATPase activity of τ was quite different from their effect on γ, the close relative of the τ subunit. The τ subunit, by itself, is a much more active DNA dependent ATPase than γ and, in fact turns over two times more ATP than the γδδ' complex. Unlike the γ ATPase, the τ ATPase was essentially unaffected by β or by δ with or without β or by δ' with or without β. However, like the γ ATPase, the presence of both δ and δ' stimulated the τATPase, although the effect was only 4-fold compared to the 30-fold stimulation of γ by δδ'. Whereas β stimulated the γδδ' ATPase 3-fold, the β subunit did not stimulate the γδδ' ATPase at all, in fact β slightly inhibited it, yet the γδδ' complex is as active as γδδ' in reconstituting a processive polymerase with β and αε.

The cloned δ' preparation appears as a doublet in a 13% SDS polyacrylamide gel and the two polypeptides are of the same size and molar ratio (2:1, lower band-to-upper band) as the δ' doublet purified from the γ complex. Electrospray mass spectometry revealed that the smaller polypeptide (δ'_(S)) was the size predicted from the gene sequence and the larger polypeptide (δ'_(L)) was increased in size by 521 Da. The nature of the larger polypeptide is presently under investigation. Possibilities include mRNA splicing, use of an upstream translational start signal, readthrough of the stop codon, translational frameshifting, and posttranslational modification. Whatever the mechanism which profuces δ'_(L) it must be efficient since the highly overproduced δ' still produces the same level of δ'_(L) relative to the δ'_(S) and δ'_(L) within the holoenzyme. Irrespective of how δ'_(L) is synthesized, the fact remains that δ'_(L) and δ'_(S) are different. Presumably they also have functional differences as in the case of the related γ and τ subunits. Whereas τ and γ both appear to be within each holoenzyme molecule, it remains to be shown whether the δ'_(L) and δ'_(S) subunits are on one or on different holoenzyme molecules.

Sequence analysis of δ'_(L) and δ'_(S) show they have identical N-termini proving δ'_(L) is not derived from an alternate upstream ATG start site. Translational readthrough of the stop codon was considered as an explanation which would produce a protein containing 19 additional amino acids before the next stop codon in the open reading frame, but this would increase the size of δ' by 2130 Da, much larger than the observed mass of δ'_(L). Treatment of δ' with calf intestinal and bacterial alkaline phosphatases did not effect the mobility of either δ'_(S) or δ'_(L) suggesting that serine and threonine phosphorylation is not involved in the formation of δ'_(L) ; attachment of other groups remains a possibility. Hence, translational frameshifting (or jumping), covalent modification (other than phosphate on Ser or Thr) and mRNA splicing remain possible.

It seems most pertinent to consider translational frameshifting as a source of δ'_(L) since such a mechanism has precedent in holoenzyme structure. The dnaX gene encoding the τ subunit of the holoenzyme generates the γ subunit by a translational frameshift into the -1 reading frame. If δ'_(L) is produced by a -1 frameshift, the frameshift would have to occur upstream of the holB stop codon but not so far upstream that a -1 frameshift would produce a truncated protein due to running into an early -1 frame stop codon. Thus the -1 frameshift would have to occur at or after the last -1 frame stop codon near Glu₃₂₀ after which translation would proceed past the normal stop codon in the open reading frame to produce a protein which is 7 amino acids larger than that predicted by the open reading frame of holB.

The γ complex expends ATP energy to clamp the β subunit onto a primer and it is this β dimer clamp that tethers the αε polymerase to the template for rapid and highly processive DNA synthesis by the αε polymerase which is only efficient after the β subunit has been clamped onto the DNA by γ complex action. A mixture of the γ and δ subunits is sufficient in this assay to clamp β onto DNA, however much more γ and δ is needed relative to the amount of γ complex. The δ' subunit stimulates γ and δ in this assay such that the amounts of γ, δ and δ' are nearly comparable with the amount of γ complex that is required (the γ and ψ subunits give another 3-8 fold stimulation of activity at low concentrations of γδδ', as described in the accompanying report. Likewise, neither δ or δ' have a large effect on the ATPase activity of γ but addition of both δ and δ' to γ gives a 30-fold stimulation of the γATPase activity. The requirement of both δ and δ' for efficient replication activity and for maximal ATPase activity of γ correlates with the physical studies in the accompanying report which show that δ and δ' form a complex and the δδ' complex binds tightly to γ, whereas when δ and δ' are added separately with γ they do not form a strong γδ or γδ' complex.

The τ subunit contains the sequence of the γ subunit (γ is produced from τ) plus an extra domain of 212 amino acids which binds to α and to DNA.

A homology search of the translated GenBank indicated that the most homologous protein to δ' of the present invention was another E. coli protein, the γ/τ subunit(s) of DNA polymerase III holoenzyme. There is 27% identity and 44% similarity including conservative substitutions over the entire length of δ' and γ/τ. One particular region in δ' of 50 amino acids (amino acids 110-159) is strikingly similar to γ/τ (amino acids 121-170) having 49% identity. A putative helix-turn-helix motif in γ/τ (Ala₁₁₄ X₃ Gly₁₁₈ X₅ Leu₁₂₄) is positioned just 19 residues downstream of the helix-turn-helix motif in δ'.

The extent of sequence homology between δ' and the γ/τ subunitr is above the level required to speculate that they have similar three dimensional structures; when both δ'_(S) and δ'_(L) are taken into account, four of the eleven subunits within the holoenzyme, according to the present invention, may have similar structures.

The interactions between δ and δ' were also studied as part of the present invention.

Equal amounts of δ and δ' were incubated together for 30 minutes at 15° C. and then analyzed by gel filtration and glycerol gradient sedimentation. Gel filtration analysis showed δ and δ' subunits comigrate and elute approximately six-to-eight fractions earlier than either δ or δ' alone indicating that they form a δδ' complex. Comparison with protein standards yields a Stokes radius of 31.1 Å. The δ and δ' also comigrated during glycerol gradient analysis and sedimented faster than either δ or δ' alone, again consistent with formation of a δδ' complex with an S value of 3.9S. Combining the S value and Stokes radius yields a native mass of 53 kDa for the δδ' complex, most consistent with the mass of a 1:1 complex of δ₁ δ'₁ (75.6 kDa) then of a higher order aggregate of δδ'. Both δ'_(L) and δ'_(S) are visible in the δδ complex indicating they are present as a mixture of δδ'_(L) and δδ'_(S). Formation of a trimeric δδ'_(L) δ'_(S) complex is unlikely as the combined mass would be 113 kDa, twice the observed mass. However, if free δ and δ' were in a rapid equilibrium with the δδ' complex then the observed mass of the δδ' complex would be a weighted average of the amount of complex and amount of free subunits and therefore the possibility of a higher order aggregate such as a δδ'_(L) δ'_(S) complex can not be rigorously excluded.

Densitometry analysis of the Coomassie Blue stained gel yielded a molar ratio of δ:δ' of 1.1:1.0, respectively (the two δ' bands were considered together as one δ') further supporting the δ₁ δ'₁ composition. Different proteins may take up different amounts of Coomassie Blue stain and therefore molar rates determined by densitometry must be regarded as tentative. A dynamic light scattering analysis of δ, δ' and δδ' complex is also presented in the table below.

The Stokes radius and sedimentation coefficient of δ, δ' and δδ' complex were determined from the gel filtration and glycerol gradient sedimentation analyses; and the native molecular mass and the frictional coefficient were calculated from the Stokes radius and S value. These calculations require the partial specific volume of δ and δ'; these volumes were calculated by summation of the partial specific volumes of the individual amino acids for each δ and δ'. Molecular weights of δ, δ' and the δδ' complex (assuming a composition of δ₁ δ'₁) were calculated from the gene sequences of δ and δ'.

    ______________________________________                                                         δ                                                                               δ' δδ'                                ______________________________________                                         Stokes radius     26.5     25.8     31.1                                       Sedimentation coefficient                                                                        3.0      3.0      3.9                                        Partial specific volume                                                                          0.74     0.74     0.74                                       Native mass (radius and S value)                                                                 34,708   33,791   52,952                                     Native mass (gene sequence)                                                                      38,704   36,934   75,630                                     Frictional coefficient                                                                           1.22     1.20     1.25                                       Diffusion coefficient (light scattering)                                                         7.60     8.16     6.61                                       Radius calculated (D)                                                                            28.2     26.3     32.5                                       ______________________________________                                    

The diffusion coefficient obtained from the light scattering analysis can be used to calculate the Stokes radius and these values were within 6% of the Stokes radius of δ, δ' and δδ' complex determined in gel filtration.

In the γ complex, the γ, δ and δ' subunits are bound together along with the χ and ψ subunits. The activity analysis described herein indicates that γ and δ interact since both are necessary and sufficient to assemble the β clamp onto DNA. Further, the δ' subunit stimulates the DNA dependent ATPase activity of γ indicating that γ and δ' interact.

The physical interaction between δ, δ' and γ were examined using the gel filtration technique which detects tightly bound protein-protein complexex, but since components are not at equilibrium during gel filtration, weak protein complexes will dissociate. The γ subunit (47 kda) is larger than δ and δ', and is at least a dimer in its native state with a large Stokes radius and quite an asymmetric shape (γ runs as a trimer or tetramer in gel filtration and as a dimer in a glycerol gradient. The γ was mixed with a 4-fold molar excess of δ and δ' then gel filtered. A complex of γδδ' was formed as indicated by the comigration of both the δ and δ' subunits with γ. The excess δδ' complex eluted much later (fraction 40-46). Since δ binds δ', it is possible that only one, for example δ, binds γ and the other (e.g. δ') is part of the complex by virtue of binding δ instead of directly interacting with γ. To determine which subunit, δ or δ', binds directly to γ, the γ subunit was mixed with either δ or δ' then gel filtered. The mixture of γ and δ showed that γ and δ' did not form a gel filterable γδ complex as indicated by the absence of δ in fractions 24-32 containing γ. The mixture of γ and δ' showed that δ' did not form a complex with γ either as indicated by the absence of δ' in fractions containing γ. Therefore both δ and δ' must be present to form a gel filterable complex with γ. Using pure cloned δ no γδ complex in gel filtration (or in glycerol gradient analysis) was seen.

The gel filtration column fractions of the γδδ' complex were analyzed for their activity in assembly of the β clamp on primed DNA. Fractions containing the γδδ' complex were quite active. The δδ' complex, even at high concentration, is not active in assembly of the β clamp and therefore the slight amount of activity in following fractions was probably due to a slight amount of γ which trailed into the peak of the δδ' complex thus giving activity in the assay. The column fractions of the γδ and γδ' mixtures were inactive except for the peak fraction of γ in the γδ' ananlysi which supported weak activity. There was a slight, barely detectable amount of δ' (but not δ), in the fractions containing γ as though a slight amount of γδ' complex was formed and survived the column.

Following these studies with δ and δ', the present invention has found that δ behaved as a monomer in gel filtration and glycerol gradient sedimentation. The δ' subunit also appeared monomeric. Neither δ or δ', when separate, formed a gel filterable complex with the γ subunit. Yet they most likely bind to γ (at least weakly) as indicated by activity assays in which γδ is active (without δ') in assembly of the β clamp, and δ' (without δ) stimulates the DNA dependent ATPase activity of γ. The δ and δ' subunits bound each other to form a gel filterable 1:1 δ'₁ δ₁ complex and when mixed with γ they efficiently formed a tight gel filterable γδδ' complex. Hence, the binding of δ and δ' to γ is cooperative.

The δ' subunit is a mixture of two related proteins, δ'_(L) and δ'_(S) which are encoded by the same gene; δ'_(L) is 521 da larger than the gene sequence predicts. The functional and structural difference between them is presently unknown. In these binding studies, both δ'_(S) and δ'_(L) bound to δ and they both assembled into the γδδ' and γδδ' complexes, consistent with the fact that both δ'_(L) and δ'_(S) are observed withion polIII and the γ complex.

No single subunit of the γ complex is active in assembling the β clamp on DNA. Presumably this reaction is to complicated for just one protein. A mixture of γ and δ is capable of assembling β onto DNA although they are inefficient and require δ' for efficient activity. Perhaps δ' increases the efficiency of γδ by physically bringing γ and δ together in the γδδ' complex, although it is also possible that δ' participates directly in the chemistry of the reaction. The γ subunit has a low level of DNA dependent ATPase activity, and described above, δ binds the β subunit. These two facts allow speculation that γ binds the primed template, and δ brings in the β subunit, then ATP hydrolysis is coupled to assemble the ring shaped β dimer around the DNA.

Since γ is known to bind ATP and has a low level of DNA dependent ATPase activity, it is an obvious candidate as the subunit which interacts with the ATP in the β clamp assembly reaction. Two molecules of ATP are hydrolyzed in the initiation reaction in which the holoenzyme becomes clamped onto a primed template to form the initiation complex. This initiation reaction has its basis in the assembly of the β clamp on DNA. The stoichiometry of two ATP hydrolyzed in formation of one initiation complex suggests two proteins hydrolyze ATP. These two proteins may be the two halves of a γ dimer. However it is also possible that δ interacts with ATP. The sequence of δ shows a very close, match to the consensus for an ATP binding site and UV induced cross-linking studies suggest that δ binds ATP. The availability of δ in quantity should now make possible a full description of the mechanism by which ATP is couplked to assemble the ring shaped β dimer around DNA.

The third subunit according to the present invention, that of θ, was also identified, purified, cloned and sequenced. N-terminal analysis of the θ peptide yielded the following sequence of 40 amino acids: ##STR17##

Based upon this sequence, two DNA probes were fashioned. These probes had the sequences of: ##STR18##

These two probes were also end-labelled with ³² P for use with Southern blot procedures.

For Southern blot analysis, E. coli DNA was cut with the 8 Kohara map enzymes [see Cell 50:495 (1987)]. The two probes described above were used to probe two Southern blots of E. coli DNA. The bands (DNA fragments) in common with the two blots were noted, as was their size. At least 3 positions on the Kohara map of the E. coli chromosome were consistent with the Southern blot fragmentation pattern.

Thus, based upon these findings, E. coli Dna digested with either EcoRV or PvuII following DNA extraction [see J. M. B. 3:208 (1961)] was run out in an agarose gel, and all the DNA in the size region of the gel corresponding to the fragment size containing e for that enzyme (PvuII or EcoRV) from the Southern blot analysis, was extracted from the gel and cloned into M13mp18 and M3mp19 using conventional techniques. The M13 transformant DNAs were analyzed by Southern blot and probed using the two probes described above. One M13 DNA was obtained with the θ sequence. When this M13 θ was sequenced, however, not all the theta gene was present; the gene extended beyond the PvuII restriction site. The M13 θ was then used as a reagent to obtain the complete θ gene.

A Kohara λ phage (λ₃₃₆) was grown and the θ gene in E. coli was excised using an EcoRV cut 2.7 kb fragment. Next, a filter containing all the Kohara λ phage was probed using the partial θ gene as the probe. Thus, it was possible to identify the λ phage containing the full θ gene.

The holE gene was then cloned from the λ phage into pUC18 and subsequently sequenced. The full genetic sequence for the θ gene was thus determined to be: ##STR19##

The open reading frame above predicts that θ is a 76 amino acid protein of 8,629 Da. The underlined nucleotide sequence exactly matches the corresponding N-terminal sequence of θ. In addition, the upstream sequence contains two putative RNA polymerase promoter signals and a Shine-Dalgarno sequence. This upstream sequence is: ##STR20##

The downstream sequence begin with a stop codon: ##STR21##

This transaction into the peptide sequence: ##STR22##

Using site-directed mutagenesis, the initial Met codon (AGA ATG) was mutated to CAT ATG (NdeI site) using an oligonucleotide with 15 bases on either side of the mutation. This was then used to obtain the overproduction of the θ gene in which the mp19θ (a 2700 bp insert) was grown in strain CJ236 cells in the presence of uridine. The purified single stranded DNA from these cells was purified and hybridized with the NdeI mutation and replicated with the holoenzyme in vitro. XLI-Blue cells were transformed with the double stranded DNA product and ten plaques were selected for miniprep sequencing; all 10 plaques contained the mutation. The θ sequence was excised from the DNA with HindIII, NdeI, and the resulting 1 kbp fragment was inserted into pET-3C [see Methods in Enzymology 185:60 (1990)]. The resulting pET-3Cθ was used to transform competent cells [BL21(DE3)]. Single colonies of the transformed cells were grown in liquid media at 37° C. to an OD of about 0.6, induced with IPTG generally as described previously, and harvested post induction. Successful overexpression of the θ peptide was obtained using this system.

The N-terminal sequence analysis of θ was examined as follows: PolIII was purified [see J. Biol. Chem. 263:6570 (1988)] except that the last step using Seperose 6 was replaced with an ATP-agarose column (Pharmacia, type II) which was eluted with a linear salt gradient. After the δδ' eluted from the ATP agarose column, a mixture of pure polIII' and γχψ complex eluted together. This mixture was separated by column chromatography on MonoQ using a linear gradient of 0-0.4M NaCl in buffer A. The polIII' which was eluted after the γχψ complex was used as the source of θ subunit. The subunit was separated from α, τ and ε subunits of polIII' by electrophoresis in a 15% SDS polyacrylamide gel, and was electroblotted (110 pmol) onto PVDF membrane. The θ subunit was visualized by Ponceau S stain, and the N-terminal sequence was determined to be: ##STR23## in which the parenthesis indicate uncertain amino acid assignments.

The θ was isolated using E. coli genomic DNA isolated from strain C600 [see J. Mol. Biol. 3:208 (1961)], cut with the Kohara panel of restriction enzymes (BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI and PvuII), and separated in a 0.8% native agarose gel. The gel was depurinated (0.25M HCl), denatured (0.5M NaOH, 1.5M NaCl) and neutralized (1M Tris, 2M NaCl, pH 5.0) prior to transfer of the DNA to Genescreen plus (DuPont New England Nuclear) using a Vacugene (Pharmacia) apparatus in the presence of 2×SSC (0.3M NaCl, 0.3M sodium citrate, pH 7.0). The membrane was air dried prior to hybridization. Two synthetic oligonucleotide DNA 57-mer probes were designed based on the N-terminal sequence of θ assuming the highest frequency of codon usage and favoring T over C in the wobble position. The two probes (5'→3') were: ##STR24##

The DNA 57-mers (100 pmol each) were 5' end-labelled using 1 μM [γ-³² P] ATP and T4 polynucleotide kinase, and then used to probe Southern blot of the restricted E. coli genomic DNA. Two Southern blots were hybridized individually using one or the other of the 57-mer probes overnight in the same buffer as above except with an additional 200 μg/ml of denatured salmon sperm DNA. The Southerns were washed in 2×SDS at room temperature for 30 minutes, then 3 hours at 42° C. (changing the buffer each hour), then exposed to X-ray film. The Theta 1 probe showed a single bnd in 7 of the 8 restriction digests; the Theta 2 probe consistently showed many bands in each lane which were eliminated equally as the hybridization and washing conditions were gradually increased in stringency, suggesting that Theta 2 did not match the true sequence of the holE gene. After holE was cloned and sequenced, it was found that 7 nucleotides of Theta 1 and 12 nucleotides of Theta 2 did match the holE sequence.

To clone the holE gene, 100 μg of E. coli DNA was digested with PvuII, and the small population of DNA fragments migrating in the 400 to 600 bp range (the Southern blot using Theta 1 probe indicated holE was on a 500 bp PvuII fragment) was extracted from the agarose gel, blunt-end ligated into M13mp18 digested with HincII, and transformed into competent XL1-Blue cells. Presence of the holE gene was determined by Southern blot analysis of minilysate DNA prepared from recombinant colonies using the 5' end-labelled Theta 1 as a probe. One positive clone was obtained and sequenced; it contained approximately one-half of the holE gene (a PvuII site lies in the middle of holE). This fragment of holE was uniformly labelled using the random primer labelling method, and used to screen the complete Kohara ordered lambda phage library of E. coli chromosomal DNA transferred onto a nylon membrane. Prehybridization and hybridization were conducted as described above except that the temperature was increased to 65° C. and the wash steps were more stringent (2×SSC, 0.2% SDS, next 1×SSC, and then 0.5×SSC at 65° C.). A single phage clone λ 19H3 (336 of the miniset) [see Cekk 50:495 (1987)] hybridized with both the genomic fragment and the Theta 1 probe.

The phage and a 2.7 kb EcoRV fragment containing the θ gene was excised, purified from a native agarose gel, and blunt-end ligated into the HincIII site of M13mp19 to yield M13mp19-θ. The 2.7 kb EcoR1-HindIII fragment from M13mp19-θ was excised, gel purified, and directionally ligated into the corresponding sites of pUC18 to generate pUC-θ. Both strands of the holE gene in pUC-θ were sequenced using the sequenase kit [α-³⁵ S]dATP, and synthetic DNA 20-mers. This time the entire holE gene was present.

An NdeI site was generated at the start codon of the holE gene by the oligonucleotide site directed mutagenesis using a DNA 33-mer: ##STR25## containing an NdeI site (underlined) at the start codon of holE to prime replication of M13mp19-θ viral ssDNA and using SSB and DNA polymerase III holoenzyme in place of DNA polymerase I. The NdeI site in the resultant phage (M13mp19-θ-NdeI) was verified by DNA sequencing. An approximately 1 kb NdeI-HindIII fragment was excised from M13mp19-θ-NdeI and directionally ligated into the corresponding sites of pUC18 to yield pUC-θ-NdeI. A 1 kb NdeI/BamHI fragment from pUC-θ-NdeI was then subcloned directionally into pET3c digested with both NdeI and BamHI to generate the overproducing plasmid, pET-θ.

Reconstitution assays contained 72 ng phage ×174 ssDNA (0.04 pmol as circles) uniquely primed with a DNA 30-mer, 0.98 SSB (13.6 pmol as tetramer) 10 ng β (0.13 pmol as dimer), and 4 ng γ complex (0.02 pmol) in 20 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 4% glycerol, 40 μg/ml BSA, 0.5 mM ATP, 60 μM dGTP, and 0.1 mM EDTA in a final volume of 25 μl (after addition of αε or αεθ). The αε and αεθ complexes were each preformed upon mixing 38 pmol each of α and ε, and when present, 152 pmol of θ in 12.5 μl of 25 mM Tris-HCl (pH 7.5), 2 mM DTT, 1 mM EDTA, 10% glycerol followed by incubation for 1 hour at 15° C. These protein complexes were diluted 30-fold in the same buffer just prior to addition to the assay on ice, then the assay tube was shifted to 37° C. for 6 minutes to allow reconstruction of the processive polymerase on the primed ssDNA. DNA synthesis was initiated upon rapid addition of 60 μM dATP and 20 νM [α-³² P]TTP, then quenched after 15 seconds and quantitated using DE81 paper. Proteins used in the resconstruction assays were purified, and their concentrations determined using BSA as a standard.

A synthetic DNA 56-mer was designed as a hooked primer template to assay 3'→5" exonuclease activity:

A DNA 56-mer (75 pmol as 56-mer) was 3' end-labelled with 75 pmol of [α-³² P] dTTP (3000 Ci/mmol) using 200 units of terminal transferase under conditions specified by the manufacturer (Boehrnger) in a total volume of 100 μl followed by spin dialysis to remove remaining free nucleotide. ##STR26##

Prior to adding proteins to the assay, θ was titrated into ε upon incubating 2 μg ε (70 pmol as monomer) with θ (0-10 μg, 0-1.16 nmol as monomer) in a total volume of 10 μl buffer A containing 50 μg/ml BSA at 15° C. for 1 hour. The εθ mixture was then diluted 100-fold using buffer A containing 50 μg/ml BSA. A 2.5 μl sample of diluted complex was added to 200 fmol 3'-³² P-end-labelled mispaired hook DNA in 12.5 μl of 25 mM Tris-HCl (pH 7.5), 4% sucrose, 5 mM MgCl₂, 8 mM DTT, and 50 μg/ml BSA followed by a 3 minute incubation at 15° C. The reaction was quenched upon spotting 13 μl of the mixture onto a DE81 filter. The amount of mispaired nucleotide remaining was quantitated, and subtracted from the total mispaired template added to obtain the amount of 3' mispaired nucleotide released.

Gel filtration was performed using HR 10/30 fast protein liquid chromatography columns, Superdex 75 and Superose 12, in buffer C. Samples containing either θ, ε or α alone, and mixtures of these subunits were incubated at 15° C. for 1 hour. The entire sample was then injected onto the column and after collection the first 5.6 ml (Superose 75) or 6.0 ml (superose 12), fractions of 160 Ml were collected and analyzed in 15% SDS polyacrylamide gels. Protein standards were a mixture of proteins of known Stokes radius and were also analyzed. Densitometry of stained gels was performed using a laser densitometer, Ultrascan XL (Pharmacia-LKB).

Subunits (α, θ, ε) alone and mixtures of these subunits were incubated 1 hour at 15° C. (with 5% glycerol), then mixed with protein standards of known S value (50 μg of each protein standard) and immediately layered onto 12.3 ml linear 10%-30% glycerol gradients in 25 mM Tris-HCl (pH 7.5), 0.1M NaCl, 1 mM EDTA. The gradients were centrifuged at 270,000×g for 44 hours (ε, θ, and εθ complex) or 26 hours (αε and αεθ) at 4° C. Fractions of 150 μl were collected from the bottom of the tube and analyzed in a 15% SDS-polyacrylamide gel stained with Coomassie Blue.

In summary, the sequence of the N-terminal 40 amine acids of θ were obtained from the θ subunit within the polIII' subassembly (αεθτ) of holoenzyme. This sequence did not match any previously identified in GenBank, and therefore the invention attempted to identify the holE gene using the Kohara restriction map of the E. coli chromosome. Two 57-mer DNA probes were made based on the N-terminal amine acid sequence of θ and were used in a Southern analysis of E. coli genomic DNA digested with the eight Kohara restriction map enzymes. One of the 57-mer probes hybridized to a single band in 7 of 8 bands obtained upon Southern analysis, indicating that these 7 fragments must overlap in the holE gene. The Kohara restriction map was searched, and four near matches were located. Since which of these positions could not be distinguished in the Kohara map as the true holE gene, the small 500 bp PvuII fragment from genomic DNA was directly cloned into M13mp18. The DNA sequence of this PvuII fragment predicted an amine acid sequence which matched exactly to the 40 residue N-terminal sequence of θ. However, this was only a partial clone of holE due to an internal PvuII site. The PvuII fragment and one of the synthetic 57-mers were subsequently used to probe the entire Kohara library of overlapping λ phage on one membrane which identified the location of holE within λ 19H3 (No. 336 of the miniset).

The Kohara restriction map of the chromosome in the vicinity of λ 19H3 shows a close match to the fragment sizes obtained from the Southern analysis. The overlapping fragments identify the position of holE at 40.4 minutes on the E. coli chromosome. DNA analysis showed two BglI sites separated by 122 bp that span the Theta 1 57-mer probe, thus explaining the absence of a BglI fragment in the Southern analysis in which a small fragment would have run off the end of the gel. This small fragment would also have been missed in the procedure used by Kohara, accounting for the single BglI site shown on the map.

A 2.7 kb EcoRI fragment was subcloned from λ 19H3 into M13mp18 and the holE gene was sequenced. The DNA predicts θ is a 76 amino acid protein of 8,647 Da, slightly smaller than the 10 kDa estimated from the mobility of θ in a SDS-polyacrylamide gel. The pl of θ based on the amino acid composition is 9.79, suggesting it is basic, consistent with its ability to bind to phosphocellulose, but not to Q Sepharose. The molar extinction coefficient of θ at 280 nm calculated from its single Trp and the two Tyr residues is 8,250 M⁻¹ cm⁻¹.

Site directed mutagenesis was performed on the holE gene cloned into M13mp18 to create an NdeI site at the initiator methionine. The holE gene was excised from the site mutated M13mp18, inserted into pUC18 (in order to use a convenient BamHI site), then a 1 kb NdeI-BamHI fragment containing holE was ligated directionally into the NdeI and BamHI sites of pET3c to yield the pET-θ overproducing plasmid in which holE expression is driven by T7 RNA polymerase. The pET-θ was introduced into BL21(DE3) cells and upon induction of T7 RNA polymerase by IPTG, θ was expressed to 63% of total cell protein. The induced subunit was freely soluble upon cell lysis and its purification was relatively straight-forward. Four liters of cells were lysed and 300 mg of pure θ was obtained in 78% overall yield after column chromatography on Q sepharose, heparin agarose, and phosphocellulose.

Specifically, the purification of θ was carried out by utilizing four liters of BL21(DE3) cells harboring the pET-θ expression plasmid were grown in 4 L of LB media containing 50 μl.ml carbenicillin. Upon growth to an OD₆₀₀ of 0.6, IPTG was added to 0.4 mM and the cells were incubated at 37° C. for 2 hours further before they were harvested by centrifugation (8.4 g wet weight) at 4° C., resuspended in 15 ml of cold 50 mM Tris-HCl (pH 7.5) and 10% sucrose, and stored at -70° C. The cells were thawed and lysed by heat lysis. The cell lysate (Fraction I, 20 ml, 880 mg) was dialyzed (all procedures were performed at 4° C.) for 2 hours against 2 L of buffer A, and then diluted 2-fold with buffer A to a conductivity equal to 50 mM NaCl. The lysate was then applied to a 55 ml Q sepharose fast flow column equilibrated in buffer A. The θ flowed through the column as analyzed by a Coomassie Blue stained 15% SDS polyacrylamide gel and confirmed by the stimulation of the ε exonuclease activity assay developed for θ. The Q sepharose flow through fraction (Fraction II, 81 ml, 543 mg) was then applied to a 50 ml column of heparin agarose (BioRad) which was equilibrated in buffer A containing 50 mM NaCl. The flow through fraction containing θ was approximately 95% pure θ(Fraction III, 110, 464 mg), and was dialyzed overnight against 2 L buffer B, then applied to a 40 ml phosphocellulose column (P11, Whatman) equilibrated in buffer B. The column was washed with buffer B and θ was eluted using a 400 ml linear gradient of 10 mM to 200 mM sodium phosphate (pH 6.5) in buffer B. Eighty fractions were collected and analyzed for θ. Fractions 42-56 were pooled (Fraction IV, 68 ml, 300 mg) and dialyzed against 2 L buffer A prior to aliquoting and storage at -70° C. The protein concentration was determined using BSA as a standard. Concentration of pure θ determined by absorbance at 280 nm using ε₂₈₀ at 8,250 M⁻¹ cm⁻¹ was 90% of the protein concentration.

    ______________________________________                                                   total            specific                                                                              fold                                                   protein total    activity                                                                              purifica-                                    Step      (mg)    units.sup.1                                                                             (units/mg)                                                                            tion   % yield                               ______________________________________                                         I   Cell Lysate                                                                              880     2.7 × 10.sup.6                                                                  3.1 × 10.sup.3                                                                  1.0    100                                 II  Q Sepharose                                                                              543     2.3 × 10.sup.6                                                                  4.2 × 10.sup.3                                                                  1.4    85                                  III Heparin   464     2.6 × 10.sup.6                                                                  5.6 × 10.sup.3                                                                  1.8    96                                      Agarose                                                                    IV  Phospho-  300     2.1 × 10.sup.6                                                                  7.0 × 10.sup.3                                                                  2.3    78                                      Cellulose                                                                  ______________________________________                                          .sup.1 One unit is defined as the increase in fmol nucleotide released pe      minute relative to the same reaction with no θ added (ε          alone).                                                                  

Throughout this description of the present invention, buffer A was 20 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, and 2 mM DTT; Buffer B was 10 mM NaPO₄ (pH 6.5), 10% glycerol, 0.5 mM EDTA, and 2 mM DTT; and Buffer C was 25 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM EDTA and 100 mM NaCl.

Studies of the purified cloned θ showed it had the same amino terminal sequence as predicted by holE (and θ within polIII' used for electroblotting), proving that the it was indeed the purified protein encoded by the cloned gene. The activity of θ (stimulation of ε) co-purified with θ throughout the preparation.

In searching for activity, the subunit was tested for polymerase activity and for endonuclease, 3'→5' exonuclease and 5'→3' exonuclease activities on ssDNA and dsDNA. However, no such activities were observed.

Since θ is one of the subunits of polIII core, it was examined for any effect it might exert on the DNA polymerase and 3'→5' exonuclease activities of α and ε. Previous work compared the ability of αε and polIII core to form the rapid and processive polymerase with holenzyme accessory proteins, but there was no significant difference between αε and the polIII core (αεθ) suggesting θ had no role in the speed and processivity of synthesis. With pure θ, assays could be performed by either adding θ to αε or omitting θ. In a comparison of the efficiency of αε complex and αεθ complex in their ability to reconstitute the rapid processive polymerase with accessory proteins, the αε (or αεθ) was mixed with the γ complex and β subunit in the presence of ATP and phage X174 ssDNA primed with a synthetic oligonucleotide and "coated" with SSB. The mixture was preincubated for 6 minutes at 37° C. to allow the γ complex time to transfer the β ring to DNA forming the preinitiation complex clamp and time for the polymerase to associate with the preinitiation complex. The rapid processive polymerase can fully replicate this template (5.4 kb) within 12 seconds. Replication was then initiated by the addition of dATP and [α-³² P]TTP, which were omitted from the preincubation, and the reaction was terminated after 15 seconds. In this assay, the effect of θ on the amount of DNA synthesis will be a reflection of either the speed or processivity of the polymerase or the binding efficiency of the polymerase to the preinitiation complex. Based on a previous comparison of as and core, θ was not expected to influence the speed or processivity of DNA synthesis. However, in the prior study, the relative affinity of αε and polIII core for the preinitiation complex was not examined.

The αε and αεθ were titrated into this reconstitution assay and the results indicate that θ had little influence in the assay. Therefore, θ does not significantly increase the affinity of αε as for the preinitiation complex. These results are also consistent with prior conclusions. The accessory protein preinitiation complex greatly stimulates the activity of the α subunit (without ε) in the reconstitution assay. However, this "α holenzyme" was half as fast as the "αε holenzyme" and is only processive for 1-3 kb. The ability of θ to stimulate this "α holenzyme" was tested in the absence of ε, but the θ subunit had no effect indicating that it did not increase the speed or processivity of the "α holenzyme" either.

θ was next examined for an effect on the 3'→5' exonuclease activity of ε using a synthetic "hooked" primer template with a 3' terminal G-T mispair. A slight (3-fold), but reproducible stimulation of θ on excision of the 3' mismatched T residue by s was observed. In the absence of ε, addition of up to 1.0 μg of θ released no 3' terminal nucleotide. These results are compatible with an earlier study comparing 3' excision rates of polIII core and αε complex in which the polIII core was approximately 3-fold faster than αε. Although a 3-fold effect is not dramatic and may not be the true intracellular role of θ, it is large enough to follow θ through the purification procedure. The stimulation of ε exonuclease activity co-purified with θ throughout the purification procedure and the overall activity was recovered in high yield.

The polIII core subassembly of the holenzyme consists of three subunits: θ, α (polymerase), and ε (3'→5" exonuclease). Gel filtration was used to analyze the ability of these individual subunits according to the present invention to assemble into the polIII core assembly. α and θ were mixed together and gel filtered; however, θ did not comigrate with α. Upon mixing ε and θ, a stable εθ complex was formed. The results of these studies are quite consistent with the activity analysis presented above in which θ had no effect on the polymerase but a noticeable effect on the activity of ε.

It has been reported that a concentrated preparation of polIII core (18 μM) was dimeric containing two molecules of polIII core which were presumed to be dimerized through interaction between their θ subunits since a concentrated solution of ae complex contained only one α and one ε. However, in the gel filtration experiments of the present invention, the reconstituted polIII core migrates only slightly larger than the α subunit indicating that θ did not act as an agent of polIII core dimerization.

In gel filtration experiments performed at a concentration of 73 μM α and 73 μM ε in either the absence of θ (αε only), the presence of a substoichiometric amount of θ (molar ratio α:ε:θ of 1:1:0.5), or with excess θ (molar ratio 1:1:3), showed that the presence of θ did not increase the aggregation state (i.e., monomer to dimer). Thus, it may be considered that the αε complex by itself is a dimer. However, comparison of αε and polIII core with size standards in the gel filtration analysis show that they elute near the 158 kDa IgG standard indicating that they are monomeric, i.e., one of each in the complex. They have a Stokes radius of 49 Å which is substantially the radius determined for the αε complex (50 Å), and similar to the 54 Å Stokes radius determined in studies of the dilute monomeric polIII core.

To increase confidence in the aggregation state of these reconstituted complexes, the study of the αε complex and reconstituted polIII core was extended to an analysis of their sedimentation behavior in glycerol gradients using the same concentration and ratio of subunits as in the gel studies. Again the αε and αεθ essentially co-sedimented regardless of whether θ was present. The αε complex and polIII core each sedimented with an S value close to that of the 150 kDa IgG size standard further indicating they are monomeric subassemblies.

The native molecular weights of θ, ε and of the ετ complex were also determined using gel filtration and glycerol gradient sedimentation. The θ and ε subunits were first analyzed separately: θ, by itself, elutes after myoglobin which is 17.5 kDa, indicating θ is a monomer (8.6 kDa) rather than a dimer of 17.2 kDa; ε migrated just after an ovalbumin standard (43.5 kDa) consistent with ε as a 28,5 monomer rather than a 57 kDa dimer.

To asses the native molecular masses of θ, ε and the εθ complex, the analysis was extended to sedimentation in glycerol gradients. The Stokes radius and S values of θ, ε and εθ complex were determined by comparison to protein standards and their observed mass was calculated. The observed masses of θ, ε and εθ are 11.6 kDa, 32.7 kDa and 35.5 kDa, respectively, values most consistent with θ as a 8.6 kDa monomer, ε as a 28.5 kDa monomer, and the εθ complex having a composition of ε₁ θ₁ (37.1 kDa); densitometric analysis of the εθ complex yielded a molar ration of 1 mol of ε to 0.8 mol θ, consistent with this composition.

The fourth subunit according to the present invention, that of ψ, was also identified, purified, cloned and sequenced. N-terminal analysis of the ψ peptide yielded a protein which, when translated to its genetic sequence was found to be identical to a portion of a much larger sequence described by Yoshikawa [see Mol. Gen. Genet. 209:481 (1987)]. However, Yoshikawa's description was for a riml sequence from E. coli responsible for encoding an enzyme catalyzing acetylation of the N-terminal portion of ribosomal protein S-18; his upstream sequencing from this gene's reading frame was purely accidental and he does not indicate any appreciation of the gene as a coding sequence for the ψ peptide.

The amino acid sequence obtained from the ψ peptide is: ##STR27##

Using the information above, the sequence was translated into the genomic structure which is: ##STR28##

In addition to the normal sequence for the genomic material, the gene also contains an internal NdeI site.

The sequence above is preceded by an upstream sequence containing two underlined RNA polymerase promoter signals (TTGGCG and TATATT), and a Shine Dalgarno (AGGAG) sequence. The complete upstream sequence is: ##STR29##

In addition, the open reading frame is followed by a downstream sequence beginning with a stop codon: ##STR30##

The ψ gene was then produced by PCR using E. coli genomic DNA and the following (5'→3') primers: ##STR31##

As can be seen, primer 1 contains a NdeI site, and primer 2 contains a BamHI site which have been underlined above.

The PCR-produced DNA was used to clone the ψ gene into pET-3c expression plasmid using a two-step cloning procedure necessitated by the internal NdeI site in the nucleic acid sequence. Briefly this procedure involved cutting the PCR product with NdeI restriction enzyme into two portions of 379 (NdeI to NdeI) and 543 (from NdeI to BamHI) bp. The 543 bp portion was ligated into plasmid pET-3c (4638 bp) to form an intermediate pET-3ca (5217 bp). The pET-3ca was then linearized, and the 379 bp portion inserted to form the desired pET-3c plasmid containing the complete PCR product insert.

The overexpression vector containing the complete insert was then inserted into E. coli, and induced with IPTG as described herein, and overexpression (an increase to over 20% of total bacterial protein) of the ψ protein was seen.

The ψ protein was purified by first dissolving the cell membrane debris in 6M urea followed by passing the resulting solutions through a hydroxylapetite column, which had been equilibrate previously with a 6M urea buffer (180 g urea, 12.5 ml 1M Tris at pH 7.5, 0.5 ml of 0.5M EDTA, and 1 ml of 1M DTT), wherein the ψ peptide will flow through while almost everything else in solution will be held within the column. The The ψ peptide outflow of the hydroxylapetite column was then bound to a DEAE column, rinsed with buffer, and eluted with a gradient of NaCl. Fractions containing the ψ peptide were pooled, dialyzed twice against 1 liter of buffer, and loaded onto a hexylamine column for final purification. Fractions from the hexylamine column containing the ψ peptide were eluted with a NaCl gradient (0.0 to 0.5M ), pooled and saved as pure ψ subunit peptide.

Studies were also conducted to determine that the ψ gene according to the present invention encodes ψ subunit peptide. These studied determined that the N-terminal analysis of native ψ peptide is predicted by the ψ gene sequence according to the present invention; native ψ peptide was obtained and digested with trypsin and a few of the resulting peptides synthesized--the sequenced peptides were encoded by the gene sequence according to the present invention; the cloned/overproduced/pure ψ peptide made in accordance with the present invention comigrated with the ψ subunit peptide within the naturally occurring holenzyme; and the ψ peptide produced from the sequence according to the present invention formed a γχψ complex when mixed with γ and χ as would occur with natural components.

The γχψ complex was purified [see J. Bio Chem. 265:1179 (1990)] from 1.3 kg of the γ/τ overproducing strain (HB101 (pNT203, pSK100). The ψ subunit was prepared from γ and χ by electrophoresis in a 15% SDS-polyacrylamide gel, then ψ was electroblotted onto PVDF membrane for N-terminal sequencing (220 pmol), and onto nitrocellulose membrane for tryptic digestion (300 pmol) followed by sequence analysis of tryptic peptides. Proteins were visualized by Ponceau S stain. The N-terminal analysis was determined to be: NH₂ TSRRDDQLQQLGIT. Two internal tryptic peptides were determined to be: ##STR32##

A 3.2 kb PstI/BamHI (DNA modification enzymes, New Endland Biolabs) fragment containing holD was excised from γ5Cl and ligated directionally into the polylinker of Blue Script (Stratagene). The 1.5 kb Accl fragment (one site is in the vector and one is in the insert) containing holD was excised, the ends filled in using Klenow polymerase, then ligated into pUC18 (cut with Accl and the end filled with Klenow) to yield pUC-ψ. Both strands of DNA were sequenced by the chain termination method of Sanger using the sequenase kit [α⁻³² P] dATP (radiochemicals, New England Nuclear), and synthetic DNA 17-mer (Oligos etc. Inc.).

A 922 bp fragment was amplified from genomic DNA (strain C600) using Vent DNA polymerase and two synthetic primers, an upstream 32-mer (CAACAGGAGCGATTC CACTATGA-CATCCCGACG), and a downstream 30-mer (GATTC GGATCCCTGCAGGCCG-GTGAATGAGT). The first two nucleotides in the NdeI site (underlined) of the upstream 32-mer and the first 11 nucleotides of the downstream 30-mer (including the underlined BamHI sequence) are not complimentary to the genomic DNA. Amplification was performed using a thermocycler in a volume of 100 μl containing 1 ng genomic DNA, 1 μM each primer, and 2.5 units of Vent polymerase in 10 mM Tris-HCl (pH 8.3), 2 mM MgSO₄, 200 μM each dATP, dCTP, dGTP and TTP. Twenty five cycles were performed: 1 minute at 94° C., 1 minute at 42° C., 2 minutes at 72° C. Amplified DNA was phenol extracted, ethanol precipitated, then digested with 50 units NdeI in 100 μl 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT and 50 mM potassium acetate (final pH 7.9), overnight at 37° C. After confirming the NdeI digestion by agarose gel, 50 units of BamHI was added and digestion was continued for 2 hours. The NdeIdeI fragment,which contained most of the holD gene, and the NdeI/BamHI fragment were separated in an agarose gel, electroeluted, phenol/chloroform extracted, ethanol precipitated and dissolved in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. The holD gone was cloned into pET3c in two steps. First the NdeI/BamI fragment encoding the C-terminus of ψ was ligated into pET3c digested with NdeI and BamHI to generate pETψ_(c-ter') (linearized with NdeI and depsphorylated) to yield the pET-ψ overproducer. DNA sequencing of the pET-ψ confirmed the correct orientation of the NdeI/NdeI fragment.

The 25 μl assay contained 72 ng M13mp18 ssDNA (0.03 pmol as circles) primed with a synthetic DNA 30-mer, 0.98 μg SSB (13.6 pmol as tetramer), 82 ng αε (0.52 pmol), and 33 ng β (0.29 pmol as dimer) in 20 mM Tris-HCl (pH 7.5), δ mM MgCl₂, 40 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 40 μg/ml BSA, 0.5 mM ATP, and 60 μM each dCTP and dGTP. Addition of χ, ψ and γδδ' complex to the assay was as follows. The γδδ' complex, χ and ψ (ψ was initially in 4M urea) subunits were preincubated before addition to the assay for 30 minutes at 4° C. at concentrations of 2.4 μg/ml γδδ' complex (14.2 nM), 0-0.75 μg/ml χ (45 nM), and 0-0.75 μg/ml ψ (0-48 nM) in 25 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 50 μg/ml BSA, 20% glycerol (buffer B) (the concentration of urea in the preincubation was 8.5 mM or less). One-half μl of this protein mixture was added to the assay (urea was 0.17 mM or less in the assay after addition of ψ) then the assay was shifted to 37° C. for 5 minutes to allow polymerase assembly before initiating DNA synthesis upon addition of dATP and [α⁻³² P] dTTP to 60 μM and 20 μM, respectively. After 20 seconds, DNA synthesis was quenched and quantitated as described in the accompanying report. Assays to quantitate θ in purification were performed likewise except the protein preincubation contained 2.4 μg/ml γδδ' (14.2 nM), 0.75 μg/ml χ (45 nM) and up to 0.25 μg/ml of protein fraction containing θ. After the 30 minute preincubation, 0.5 μl was added to the assay reaction. The SSB, α, ε, β, γ, and τ subunits used in these studies were purified, and the χ, δ and δ' subunits were prepared from their respective overproducing strains. Concentrations of β, δ, δ', χ and ψ were determined from their absorbance at 280 nm using their molar extinction coefficients: β, 17,900 M⁻¹ cm⁻¹ ; δ, 46,137 M⁻¹ cm⁻¹ ; δ', 60,136 M⁻¹ cm⁻¹ ; χ, 29, 160 M⁻¹ cm⁻¹ ; and ψ 24,040 M⁻¹ cm⁻¹.

The μl assay contained 140 ng M13mp18 ssDNA in 25 mM Tris-HCl (pH 7.5), and mM MgCl₂, 50 μM [γ³² -P]ATP, 5.45 pmol γ or τ (as dimers), 10.9 pmol χ and/or ψ (as monomers) (unless indicated otherwise) and 1.4 μg SSB (19.4 pmol as tetramer) (when present). Mixtures of proteins (ψ was initially 2 mg/ml (0.13 mM ) in 4M urea) were preincubated 30 minutes on ice at 3.8 μM of each subunit (as monomer) in 30 μl of 25 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 20% glycerol (0.1M urea final concentration) before addition to the assay (15 mM urea final concentration). Assays were incubated at 37° C. for 60 minutes 5 minutes for assays containing τ) then quenched upon spotting 0.5 μl on polyethyleneimine thin layer plates (Brinkman Instruments Co.). After chromatography in 0.5M LiCl, 1M formic acid, the free phosphate at the solvent front and ATP remaining near the origin were quantitated by liquid scintillation,

Samples of ψ (45 μg, 3 nmol as monomer (initially in 4M urea)), or a mixture of ψ (45 μg, 3 nmol as monomer) with either γ (65 μg, 0.7 nmol as dimer) or τ (98 μg, 0.7 nmol as dimer) were incubated in 200 μl 25 mM Tris-HCl (pH 7.5), 0.1M NaCl, 1 mM EDTA, 10% glycerol (0.5M urea was present after addition of ψ) for 30 minutes at 15° C. The 200 μl sample was injected onto a Pharmacia HR 10/30 gel filtration column of either Superdex 75 or Superose 12 at a flow rate of 0.35 ml/min in 25 mM Tris-HCl (pH 7.5)° 0.1M NaCl, 1 mM EDTA, 10% glycerol. After the first 5.6 ml, fractions of 170 pl were collected and analyzed in a 15% SDS polyacrylamide gel and the value of Kav was calculated.

A sample of ψ (45 μg, 3 nmol as monomer, initially in 4M urea) in 200 μl 25 mM Tris-HCl (pH 7.5), 0.1M NaCl, 1 mM EDTA, 5% glycerol (0.5M urea final concentration after addition of ψ) was layered onto a 12.3 ml gradient of 10%-30% glycerol in 25 mM Tris-HCl (pH 7.5), 0.1M NaCl, 1 mM EDTA. Protein standards in 200 μl of the same buffer were loaded in another tube and the gradients were centrifuged at 270,000×g for 44 hours at 4° C. Fractions of 150 μl were collected and analyzed in a 15% SDS polyacrylamide gel stained with Coomassie Blue.

The γδδ' complex was formed upon incubation of 60 μg δ (1.55 nmol as monomer) and 60 μg δ' (1.62 nmol as monomer) with an excess of γ (600 μg, 6.4 nmol as dimer) for 30 minutes at 15° C. in 1 ml of 25 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol (buffer A). The mixture was chromatographed on a 1 ml HR 5/5MonoQ column, and eluted with 30 ml linear gradient of 0M-0.4M NaCl in buffer A. The γδδ' complex eluted at an unique position, after the elution of free δ', δ and γ (in that order) and was well resolved from the excess γ. The pure γδδ' complex was dialyzed against buffer A to remove salt. Protein concentration was determined using BSA as a standard. Molarity of γδδ' was calculated from protein concentration assuming the 170 kDa mass of a complex with subunit composition γ₂ δ₁ δ'₁, the composition expected from stoichiometry of subunits in the γ complex.

The γχψ complex was prepared from 1.3 kg of E. coli and the ψ subunit was resolved from the γ and χ subunits in a SDS-polyacrylamide gel, then electroblotted onto PVDF membrane for analysis of the amino acid sequence of the amino terminus of ψ. The ψ was also electroblotted onto nitrocellulose followed by tryptic digestion, HPLC purification of peptides and sequence analysis of two tryptic peptides. Search of the GenBank for DNA sequences encoding these peptides identified a sequence which was published in a study of the riml gene [see Mol Gen Genet 209:481 (1987)]. In order to define the operon structure of this DNA, the DNA upstream of riml was sequenced. All three peptide sequences of ψ were in one reading frame located immediately upstream of riml at 99.3 minutes on the E. coli chromosome which putatively encodes ψ and referred to as holD.

The promoter for holD underlined in the sequence has been identified previously as the promoter for the riml gene, encoding the acetylase of ribosomal protein S18, which initiates 29 nucleotides inside of holD. Hence, holD is in an operon of riml. Production of ψ was inefficient relative to ριμI protein as judged by the maxicell technique which detected riml protein but not ψ. The promoter measured by Northern analysis was strong [see Mol Gen Genet 209:481 (1987)] and the Shine-Dalgarno sequence is a good match to the consensus sequence, as is the spacing from the ATG needed for sufficient translation. Although the cellular abundance of ψ is not known, if one assumes all the ψ sequestered within the holenzyme, then it is present in very small amounts, there being only 10-20 copies of the holenzyme in a cell. Perhaps the 3-11 fold more frequent use of some rare codons may contribute to inefficient translation (Leu (UUA), Ser (UCA and AGU), Pro (CCU and CCC), Thr (ACA), Arg (CGA and CGG)).

The open reading frame of holD encodes a 137 amino acid protein of 15,174 Da. Amino terminal analysis of the ψ protein within the γχψ complex showed it was missing the initiating methionine. The molar extinction coefficient of ψ calculated from its 4 Trp and 1 Tyr is 24,040 M⁻¹ cm⁻¹. There is a potential for a leucine zipper at amino acid residues 25-53, although three prolines fall within the possible leucine zipper. There is also a helix-turn-helix motif (A/GX₃ GX₅ I/V) at G₂₂ G₂₆ I₃₃, but again prolines may preclude helix formation. There is no apparent nucleotide binding site or zinc finger motif.

The polymerase chain reaction was used to amplify holD from genomic DNA. The synthetic DNA oligonucleotides used as primers were designed such that an NdeI site was formed at the initiating ATG of holD and a BamHI site was formed downstream of holD. The amplified holD gene was inserted into the NdeI/BamHI sites of pET3c in two steps to yield pET-ψ in which holD is under control of a strong T7 promoter and is in a favorable context for translation. The sequence of holD in pET-ψ was found to be identical to that depicted in the sequence, and transformation into BL21(DE)plysS cells and subsequent induction of T7 RNA polymerase with IPTG, the ψ protein was expressed to approximately 20% of total cell protein.

The ψ protein was completely insoluble and resisted attempts to obtain even detectable amounts of soluble ψ (lower temperature during induction, shorter induction time, and extraction of the cell lysate with 1M NaCl were tested); it was necessary to resort to solubilization of the induced cell debris with 6M urea followed by column chromatography in urea. The ψ was approximately 40% of total protein in the solubilized cell debris and was purified to apparent homogeneity upon flowing it through hydroxyapatite, followed by column chromatography on DEAE sepharose and heparin agarose. By this procedure, 22 mg of pure ψ was obtained from 1 liter of cell culture in 61% yield. The pure W remained in solution upon complete removal of the urea by dialysis as described in greater detail below.

Four liters of E. coli cells (BL21(DE3)plysS) harboring the pET-ψ plasmid were grown at 37° C. in LB media supplemented with 1% glucose, 10 μg/ml thiamin, 50 μg/ml thymine, 100 μg/ml ampicillin and 30 μg/ml chloramphenicol. Upon reaching an OD of 1.0, IPTG was added to 0.4 mM and after an additional 2 hours of growth at 37° C., the cells were harvested by centrifugation (20 g wet weight), resuspended in 20 ml of 50 mM Tris-HCl (pH 7.5) and 10% sucrose (Tris-sucrose) and frozen at -70° C. The cells lysed upon thawing (due to lysozyme formed by the plysS plasmid), and the following components were added on ice to pack the DNA and precipitate the cell debris: 69 ml Tris-sucrose, 1.2 ml unneutralized 2M Tris base, 0.2 ml 1M DTT, and 9 ml of heat lysis buffer (0.3M spermidine, 1M NaCl, 50 mM Tris-HCl (pH 7.5), 10% sucrose). After 30 minutes incubation on ice, the suspension was centrifuged in a GSA rotor at 10,000 rpm for 1 hour at 4° C. The cell debris pellet was resuspended in 50 ml buffer B using a dounce homogenizer (B pestle), then sonicated until the viscosity was greatly reduced (approximately 2 minutes total in 15 second intervals) and centrifuged in two tubes at 18,000 rpm in a SS34 rotor for 30 minutes at 4° C. The pellet was resuspended in 50 ml buffer B containing 1M NaCl using the dounce homogenizer, then pelleted again. This was repeated, and followed again by homogenizing the pellet once again in 50 ml buffer B and pelleted as was done initially. The following procedures were at 4° C. using only one-fourth of the pellet (equivalent to 1 liter of the cell culture). The assay for ψ is described above, and column fractions were analyzed in 15% SDS polyacrylamide gels to directly visualized the ψ protein and aid the exclusion of contaminants during the pooling of column fractions. The pellet was solubilized in 25 ml buffer A containing freshly deionized 6M urea. The solubilized pellet fraction (fraction I, 85 mg, 22 ml) was passed over a 10 ml column of hydroxyapatite and equilibrated in buffer A plus 6M urea. The ψ quantitatively flowed through the hydroxyapatite column giving substantial purification. The protein which flowed through the hydroxyapatite column was immediately loaded onto a 10 ml column of DEAE sephacel, equilibrated in buffer A containing 6M freshly deionized urea, and eluted with a 100 ml gradient of 0-0.5M NaCl in buffer A containing 6M freshly deionized urea over a period of 4 hours. Fractions of 1.25 ml were collected and analyzed for ψ as described. Fractions were pooled and dialyzed overnight against 2 liters of buffer A containing 3M freshly deionized urea and then loaded onto a 10 ml column of hexylamine sepharose. The hexylamine column was eluted with a 200 ml gradient of 0M-0.5M NaCl in buffer B containing 3M freshly deionized urea over a period of 4 hours. Eighty fractions were collected (2.5 ml each) and were analyzed for ψ, then fractions containing ψ were pooled (fraction IV, 21.6 mg) and urea was removed by extensive dialysis against 25 mM Tris-HCl (pH 7.5), 0.1M NaCl, 0.5 mM EDTA (3 changes of 2 liters each). Protein concentration was determined using BSA as a standard, except at the last step in which a more accurate assessment of concentration was performed by absorbance using the value ε₂₈₀ equal 24,040 M⁻¹ cm⁻¹ calculated from the sequence of holD. After the absorbance measurement, DTT was added back to 5 mM and the ψ was aliquoted and stored at -70° C.

    ______________________________________                                                   total             specific                                                                              fold                                                  protein total     activity                                                                              purifica-                                                                             %                                    Fraction  (mg)    units.sup.1                                                                              (units/mg)                                                                            tion   yield                                ______________________________________                                         I   Solubilized                                                                              85.0    104.7 × 10.sup.7                                                                 12.0 × 10.sup.6                                                                 1.0    100                                    pellet                                                                     II  Hydroxy-  42.5    95.9 × 10.sup.7                                                                  22.6 × 10.sup.6                                                                 1.8    92                                     lapatite                                                                   III DEAE      30.6    89.7 × 10.sup.7                                                                  29.3 × 10.sup.6                                                                 2.4    86                                     Sepharose                                                                  IV  Hexlyamine                                                                               21.6    63.9 × 10.sup.7                                                                  29.6 × 10.sup.6                                                                 2.4    61                                     Sepharose                                                                  ______________________________________                                          .sup.1 One unit is defined as pmol of nucleotide incorporated in one           minute over and above the pmol incorporated in the assay in the absence o      added                                                                    

The pure γ protein comigrated with the y subunit of polIII* (holenzyme lacking only β) in a 15% SDS-polyacrylamide gel. Analysis of the N-terminal sequence of the pure cloned ψ matched that of the holD sequence and the sequence of the natural ψ from within the γχψ complex indicating that the purified protein encoded by the gene had been cloned.

The pure ψ appeared fully soluble in the absence of urea. However, a 2 mg/ml solution of ψ which appeared clear, and could not be sedimented in a table top centrifuge, still behaved as an aggregate in a gel filtration column. Therefore, even though ψ appeared soluble it was still an aggregate. The aggregated ψ had only weak activity in the replication assay and was inefficient in binding to other proteins in physical studies. Therefore before using ψ in assays or in physical binding experiments, urea was added to a concentration of 4M to disaggregate ψ. Once disaggregated, the urea could be quickly removed by gel filtration and ψ behaved well during filtration in the absence of urea in the column buffer. However, upon standing a full day at high concentration (>1 mg/ml) in the absence of urea, it would aggregate again. ψ would work in urea provided the preparation was sufficiently concentrated 2 mg/ml) such that it could be diluted at least 8-fold (to 0.5M urea) for protein-protein interaction studies, 300-fold for ATPase assays, and 30,000-fold for replication assays. In 0.5M urea, the ψ bound to γ and τ, and also to the χ subunit. ψ treated in this manner was also functional in stoichiometric amounts with other proteins in replication and ATPase assays.

In a previous study, a γχψ complex was purified by resolving the δ and δ' subunits out of the γ complex leaving only a complex of γχψ. Compared to γ, this γχψ complex was approximately 3-fold more active in reconstituting the processive polymerase with δ, β, and αε at elevated salt concentration. The simplest explanation for this result is that at elevated salt, γχψδ is more active than γδ in assembling the β ring around primed DNA.

The present invention indicates that a mixture of the γ, δ δ' subunits formed a stabile (gel filterable) γδδ' complex when the αε complex and β subunit were incubated with the γδδ' complex (with or without χ and/or ψ) in a reaction containing SSB "coated" M13mp18 ssDNA primed with a synthetic DNA oligonucleotide and in the presence of 40 mM added NaCl, and the reaction was incubated at 37° C. for 5 minutes to allow the accessory proteins time to assemble the preinitiation complex clamp and for the a αε to bind the preinitiation complex (the preinitiation complex is known to consist of a β dimer ring clamped onto the DNA). Replication of the circular DNA was then initiated upon addition of the remaining dNTPs and was quenched after 20 seconds, sufficient time for the rapid and processive holenzyme to complete the circle.

The results indicated that as ψ is titrated into the assay the replication activity increased approximately 3.5-fold and plateaued at approximately 1 mol ψ (as monomer) per mol γδδ' complex. ψ (without χ) stimulates γδδ' and χ does not stimulate the reaction, but the presence of both χ and ψ yields the most synthesis as though χ does exert an influence on the assay but only when ψ is also present.

Previously γ was observed to contain a low level of DNA dependent ATPase activity (0.1 mol ATP hydrolyzed/mol γ/minute) compared to the ATPase of the γ complex (6.8 mol ATP/mol γ complex/minute). The γχψ complex resolved out of the γ complex appeared to contain approximately 3-4 fold more DNA dependent ATPase activity than γ suggesting that χ and/or ψ stimulated the ATPase activity of γ, or that there was an ATPase activity inherent within χ and/or γ. Now that the holC and holD genes have made available pure χ and ψ in quantity, they have been studied studied them for ATPase activity and for their effect on the DNA dependent ATPase activity of γ.

As part of these studies of ATPase activity, all possible combinations of χ, ψ and ψ have been tested. These assays were performed in the presence of M13mp18 ssDNA, one of the best DNA effectors in the previous study of the γ complex ATPase activity. The results showed that ψ alone, χ alone, and a mixture of χ and ψ had no detectable ATPase activity and therefore neither ψ nor χ would appear to have an intrinsic ATPase activity, although on the basis of negative evidence we can not rule out the possibility of a cryptic ATPase; the γ subunit has a weak ATPase activity. The χ subunit has no effect on the ATPase activity of γ. However, addition of ψ to γ stimulated the ATPase activity of γ approximately 3-fold. Titration of ψ into the ATPase assay showed ψ saturated the ATPase assay at approximately 2 mol ψ (as monomer) to 1 mol γ (as dimer). Addition of the χ subunit to the γψ mixture resulted in a further 30% increase in ATPase activity.

In the presence of SSB which "coats" the ssDNA, the ATPase activity of γ, γψ and γχψ were all greatly reduced (50-fold). However, of the remaining activity, the γχψ complex was 4-fold more active than γψ showing that χ significantly stimulates the γψ ATPase which the DNA is "coated" with SSB.

The ATPase assay of ψ and χ was extended to the DNA dependent ATPase activity of the τ subunit. The τ and γ subunits are encoded by the same gene and, as a result, τ contains the γ sequence plus approximately another 24 kDa of protein which is responsible for both the ability of τ to bind DNA and to bind the polymerase subunit, α. In addition, τ has a much greater DNA dependent ATPase activity than γ, approximately 6-10 mol ATP hydrolyzed/mol τ/minute for a 60-fold greater activity of τ relative to γ.

Neither ψ, χ, or a mixture of χ and ψ had a significant influence on the ATPase activity of τ. "Coating" the ssDNA with SSB reduced the ATPase activity of τ 20-fold, and now the χ and ψ subunits stimulated the τ ATPase 10-fold to bring its activity back to about half of its value in the absence of SSB. In this case, with SSB present, the ψ stimulated τ approximately 3-fold, and χ, which had no effect on τ without ψ, stimulated the γψ ATPase another 3-fold.

To gain a better understanding of the ψ molecule the present invention studied the hydrodynamic properties of ψ in gel filtration and glycerol gradient sedimentation to determine whether ψ is a monomer or a dimer (or larger). The Stokes radius of ψ was 19 Å upon comparing its position of elution from a gel filtration column with that of protein standard of know Stokes radius. The ψ eluted in the same position as myoglobin (17.5 kDa) indicating ψ is a 15 kDa monomer rather than a dimer of 30 kDa. The ψ protein sedimented with an S value of 1.95 relative to several protein standards, and was slightly slower than myoglobin which is consistent with ψ as a monomer. If a protein has an asymmetric shape, its migration will not reflect its true weight in either of these techniques. However the effect of asymmetric shape has opposite effects in these techniques and can be eliminated by the fact that the shape factor cancels when the S value and Stokes radius are both combined in one mass equation. This calculation results in a native molecular mass for ψ of 15.76 kDa, close to the 15 kDa monomeric mass of ψ calculated from its gene sequence. Hence ψ behaves as a monomer under these conditions. The frictional coefficient of ψ calculated from its Stokes radius and native mass is 1.13, slightly greater than 1.0 which indicates some asymmetry in the shape of ψ.

Although the initial use of 4M urea would have monomerized ψ if it were a native dimer, the ψ preparation was diluted such that the concentration of urea was 0.5M before it was applied to either the gel filtration column or the glycerol gradient, and the buffer used in the column and in the gradient contained no urea. Of course, one should still be concerned that 0.5M urea is high enough to disaggregate a dimer of ψ and that the dimer hasn't time to reassociate during filtration and sedimentation. Yet under these very conditions it was found that ψ forms a protein-protein complex with γ, with τ and also with χ. Therefore it seems likely that if ψ were naturally a dimer, that the dimer could have reformed under these same conditions under which ψ can bind all these other subunits. Further, a monomeric nature of ψ is not unusual as most subunits of the holenzyme are monomers when isolated (α, ε, θ, χ, δ, δ', (only γ, τ and β are dimers).

A complex of γχψ can be purified from cells indicating that ψ or χ (or both) must directly interact with γ.

Gel filtration of a mixture of γ with a 4-fold molar excess of ψ showed that ψ coeluted with the γ subunit followed later by the elution of the rest of the excess ψ. Hence, the ψ subunit does in fact bind directly to γ.

The fifth subunit according to the present invention, χ, began with the N-terminal analysis of χ which provided a sequence a portion of which, was found to have been related, in part, to the sequence of the xerB gene [see EMBO 8(5):1623 (1989)]. Although not included in the 1692 bp sequence in the publication, a fuller more complete sequence (from 1 to 2035) of the xerB gene was provided to GenBank. In this submission, the "front-end" portion of the χ gene according to the present invention was presented. However, in neither the publication nor in GenBank was the "front-end" portion as coding for a protein. Based upon the molecular weight of χ as determined in a SDS-PAG gel analysis, the "front-end" portion reported in GenBank predicts approximately 70% of the expected length of χ.

A subsequent literature study located a gene named valS which was located downstream of the xerB gene. It appeared (and was confirmed during the research leading to the present invention) that the χ, in its entirety, must be located between the xerB and valS genes.

Edman degradation amino acid sequencing was performed on an Applied Biosystems 470A gas phase microsequencing apparatus. The γχψ complex of the holenzyme was purified, and 10 μg was electrophoresed in a 15% polyacrylamide gel [see Nature 227:680 (1970)] followed by transfer to an Immobilon membrane PVDF (Millipore) for N-terminal sequence analysis as with the previous subunits according to the present invention. Internal peptide sequences were obtained by electrophoresis of 10 μg of the γχψ complex in 15% polyacrylamide gel, followed by transfer to nitrocellulose membrane, digestion by trypsin in situ, and analysis by gas phase microsequencing.

The χ or holC gene, according to the present invention, is located at 96.5 minutes on the E. coli chromosome and encodes a 147 amino acid protein of 16.6 kDa.

The recombinant λ phage 5C4 from the overlapping λ library of Kohara [see Cell 50:495 (1987)] was used in determining the DNA sequence of the χ gene. The DNA fragment containing the χ gene was identified and cloned into pUC18 using conventional techniques. The DNA sequence for both strands of the χ gene were performed on the dublex plasmid by the dideoxy chain termination method of Sanger using the Sequenase kit; sequencing reactions were analyzed on 6% polyacrylamide, 50% (w/v) urea gels.

The sequence of the primers (5'→3') used for PCR amplification of the χ gene during the cloning of the χ gene are as follows: ##STR33##

The 30-mer hybridizes over the initiation codon of χ, and a two nucleotide mismatch results in a NdeI restriction site (underlined) at the ATG initiation codon upon amplification of the gene. The 28-mer anneals within the valS gene downstream of the c gene; a BamHI restriction site (underlined) is embedded within the six nucleotides which do not hybridize to valS.

Using these codons, subsequent studies isolated the c gene sequence which is, according to the present invention: ##STR34##

The upstream portion of the holC gene is: ##STR35##

The downstream sequence begins with the stop codon: ##STR36##

The underlined nucleotide sequences indicate the potential Shine-Dalgamo sequence (AAGAAG) of holC and the nearest possible promotor signals (TTGCCG and TATCCG) are highlighted in the first underlined region. The stop codon of the upstream XerB gene (TAA) and the start codon of the downstream ValS gene (ATG) are each double underlined.

This translated into the correct peptide which is: ##STR37##

EXAMPLE IV

(molecular cloning, cell growth and purification)

PCR reactions were performed with both Vent polymerase (Biolabs) and Taq polymerase. In a 100 μl volume, the PCR reaction was conducted in a reaction buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, and 0.01% (w/v) gelatin, 1.0 μM of each primer, and 200 μM each dNTP (Pharmacia-LKB), on 1 ng E. coli genomic DNA (prepared from K12 strain C600) with 2.5 u polymerase. PCR amplification was performed in a DNA Thermal cycler model 9801 using the following cycle: melting temperature 94° C. for 1 min, annealing temperature 60° C. for 2 min, and extension temperature 72° C. for 2 min. Alter 30 cycles, the amplified DNA was purified by phenol extraction in 2% SDS followed by sequential digestion of 10 μg DNA with 10 u NdeI, followed by 10 u BamHI. The 600 bp DNA fragment was purified from a 0.8% agarose gel by electroelution, and ligated into pET3c previously digested with both NdeI and BamHI restriction enzymes. The resulting plasmids (pETχ-1, pETχ-2 and pETχ-3) were ligated into E. coli strain BL21(DE3)pLysS.

The freshly transformed BL21(DE3)pLysSpETχ cells were grown in LB media containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol at 37° C. Upon growth to an OD₆₀₀ of 0.7, isopropyl B-D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM. Incubation was continued for 3 hr at 37° C. before harvesting the cells.

Seven mg of homogeneous χ protein was purified from a 4-liter induced culture in which nearly 30% overproduced χ protein was in soluble form. The 4-liter culture was grown in an OD₆₀₀ of 0.7 before addition of IPTG to 0.4 mM. After a further 3 hr incubation at 37° C., the cells (25 g) were harvested, resuspended in 25 ml ice-cold 50 mM Tris/10% sucrose, and lysed by 25 mg lysozyme on ice for 45 min and a subsequent incubation at 37° C. for 5 min in 5 mM Tris, 1% sucrose, 30 mM spermidine, and 100 mM NaCl. The cell lysate was clarified by centrifugation at 12,000 rpm for 1 hr at 4° C. All subsequent column chromatography procedures were at 4° C. All the columns were equilibrated in buffer A (20 mM Tris (pH 7.5), 0.5 mM EDTA, 5 mM DTT, and 20% glycerol). The χ protein was followed through the purification process by SDS-PAGE gel analysis. Total protein was estimated [see Anal. Biochem 72:248 (1976)] using bovine serum albumin as a standard. The soluble lysate (120 ml, 520 mg protein) was dialyzed against 4 liter buffer A for 16 hours before being loaded onto a 60 ml heparin-agarose column. The fractions containing χ, which eluted off the column during wash with buffer A, were pooled (360 ml, 365 mg protein), and loaded directly onto a FPLC 26/10 Q sepharose fast flow column. The Q sepharose fast flow column was eluted with a 650-ml linear gradient of 0M to 0.5M NaCl in buffer A. The fractions containing χ, eluted at approximately 0.16M salt in a volume of 45 ml (60 mg protein), were pooled, dialyzed overnight against 4 liter buffer A, and loaded onto a 1 ml N-6 ATP-agarose column. The γ complex (γδδ'χψ) binds to the column tightly due to the strong ATP binding capacity of γ, while χ protein by itself flows through. This column was included to eliminate any γ complex from the χ preparation.

The flow-through of the ATP-agarose column was loaded onto an 8 ml hexylamine column and χ was eluted with an 80 ml linear gradient of 0 to 0.5M NaCl in buffer A. The χ protein eluted at approximately 0.25M salt. Fractions containing the peptide (81 ml, 36 mg protein) were pooled and dialyzed against buffer A. The χ protein was loaded onto an 8 ml FPLC Mono Q column, and eluted with a 80 ml linear gradient of 0 to 0.5M NaCl in buffer A. The fractions containing χ (28 ml 8.5 mg protein) eluted sharply at 0.16M salt. The χ protein was pooled and dialyzed overnight against 4 liters of buffer A, then aliquoted and stored at -70° C.

The concentration of purified χ protein was determined from its absorbance at 280 nm. The molar extinction coefficient at 280 nm (ε₂₈₀) of a protein in its native state can be calculated from its gene sequence to within ±5% by using the equation ε₂₈₀ =Trp_(m) (5690 M⁻¹ cm⁻¹)+Tyr_(n) (1280 M⁻¹ cm⁻¹) [see Analytical Biochemistry 182:319 (1989)] wherein m and n are the numbers of tryptophan and tyrosine residues, respectively, in the peptide. The molar extinction coefficients of tryptophan and tyrosine are known [see Biochemistry 6:1948 (1967)]. For χ protein where m equals 4 and n equals 5, ε₂₈₀ =29,160 M⁻¹ cm⁻¹. The χ protein was dialyzed against buffer A containing no DTT prior to absorbance measurement. SDS-PAG was in 15% polyacrylamide 0.1% SDS gel in Tris/glycine/SDS buffer [see Nature 227:680 (1970)]. Proteins were visualized by Coomassie Brilliant Blue Stain.

The γχψ complex was purified from 1.3 kg of E. coli The χ subunit was resolved from the γ and ψ subunits upon electorphoresis in a 15% SDS polyacrylamide gel followed by transfer of χ onto PVDF membrane for N-terminal sequence analysis (210 pmol), and onto nitrocellulose membrane for tryptic analysis (300 pmol). Proteins were visualized by Ponceau S stain. The amino terminus of c was determined to be: ##STR38## wherein Xxx is an unidentified residue. Tryptic digestion and analysis of four internal peptides were determined to be: ##STR39##

The 3.4 kb BamHI fragment containing holC was excised from γ 5C4 and ligated into the BamHI site of pUC-χ. Both strands of the holC gene were sequenced on the duplex plasmid by the chain termination method of Sanger, and synthetic 17-mer DNA oligonucleotides. Sequencing reactions were analyzed on 6% (w/v) acrylamide, 50% (w/v) urea gels and were performed with both dGTP and DITP.

The sequences of the primers used to amplify the holC gene were: ##STR40## The upstream 30-mer hybridizes over the initiation codon of holC, and a two-nucleotide mismatch results in a NdeI restriction site (underlined above) at the ATG initiation codon upon amplification of the gene. The downstream 28-mer sequence within the valS gene downstream of holC. A BamHI restriction site (underlined) is embedded in the sequence resulting in three nucleotides which are not complementary to valS. Amplification reactions contained 1.0 μM of each primer, 200 μM of each dNTP, 1 ng E. coli genomic DNA (from strain C600), and 2.5 units of Taq I DNA polymerase in a final volume of 100 μl 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, and 0.01% (w/v) gelatin. Amplification was performed in a thermal cycler using the following cycle: 94° C., 1 minute; 60° C., 2 minutes; and 72° C., 2 minutes. After 30 cycles, the amplified 604 bp DNA was purified by phenol extraction in 2% SDS followed by sequential digestion of 10 μg DNA in 10 units of NdeI and then 10 units of BamHI according to manufacturer's specifications. The NdeI-BamHI fragment was electroeluted from a 0.8% agarose gel and ligated into gel purified pET3c previously digested with both NdeI and BamHI. The resulting plasmid, pET-χ was sequenced which confirmed that no errors had been introduced during amplification, and it was then transformed into strain BL21(DE3)plysS.

The γ subunit was purified from an overproducing strain, and the δ, δ' and ψ subunits were purified from their respective overproducing strains as described above. A mixture of 48 μg γ (0.51 nmol as dimer), 144 μg δ (3.7 nmol as monomer}, 144 μg δ' (3.9 nmol as monomer), and 192 μg ψ (12.7 nmol as monomer) was incubated at 15° C. for 1 hour and then loaded onto a 1 ml HR 5/5Mono Q column. The concentration of γ was determined using BSA as a standard. Concentrations of δ, δ' and ψ were determined from their absorbance at 280 nm using the molar extinction coefficients 46,137 M⁻¹ cm⁻¹, 60,136 M⁻¹ cm⁻¹, and 24040 M⁻¹ cm⁻¹, respectively. The column was eluted with a 32 ml gradient of 0M-0.4M NaCl in 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, and 20% glycerol (buffer A) whereupon the γδδ'ψ complex resolved from uncomplexed subunits by eluting later than all the rest. Eighty fractions were collected and analyzed by a Coomassie Blue stained 15% SDS polyacrylamide gel. Fractions containing the γδδ'ψ complex, were pooled, the protein concentration was determined using BSA as a standard, and then the γδδ'ψ complex was aliquoted and stored at -70° C. Molarity of γδδ' was calculated from the protein concentration assuming the 185 kDa mass calculated from gene sequences assuming a stoichiometry of γ₂ δ₁ δ'₁ ψ₁ as expected from the tentative stoichiometry of subunits in the γ complex.

The reconstitution assay contained 72 ng M13mp18 ssDNA (0.03 pmol as circles) uniquely primed with a DNA 30-mer, 980 ng SSB (13.6 pmol as tetramer), 10 ng β (0.13 p mol as dimer), 55 ng αε complex (0.35 pmol) in a final volume (after addition of proteins) of 25 μl 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, mM MgCl₂, 5 mM DTT, 4% glycerol, 40 μg/ml BSA, 0.5 mM ATP, 40 mM NaCl, 60 μM each dCTP, dGTP, dATP, and 20 μM [α-³² P]. Pure χ protein or column pool containing χ (1-12 ng) was preincubated on ice for 30 minutes with 37 ng γδδ'ψ complex (0.2 pmol) in 20 μl of 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol, and 50 μg/ml BSA before dilution with the same buffer such that 0.14 ng (0.76 fmol) of the γδδ'ψ complex was added to the assay in a 1-2 μl volume. The assay was then shifted to 37° C. for 5 minutes. DNA synthesis was quenched by spotting directly onto DE81 filter paper and quantitated. The αε complex, β and SSB proteins used in the reconstitution assay were purified and their concentrations determined using BSA as a standard except for β which was determined by absorbance using an ε₂₈₀ value of 17,900 M⁻¹ cm⁻¹.

Gel filtration analysis was performed using the Pharmacia HR 10/30 fast protein liquid chromatography columns, Superdex 75 and Superose 12. Proteins were incubated together for 1 hour at 15° C. in a final volume of 200 μl buffer B (25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, and 100 mM NaCl). The ψ protein was first brought to 4M in urea to disaggregate it, and when present with other proteins the final concentration of urea in buffer B was 0.5M. The entire sample was injected, the column was developed with buffer B, and after collecting the first 6 ml, fractions of 170 μl were collected. The χ protein was located in column fractions by analysis in 15% SDS-polyacrylamide gels. Densitometry of Coomassie Blue-stained gels was performed using a laser densitometer (Ultrascan XL).

Individual samples of χ (46 ng, 2.8 nmol as monomer) and of ψ (45 ng, 3 nmol as monomer), and a mixture of χ (218 ng, 13 nmol as monomer) and ψ (45 ng, 3 nmol as monomer) were incubated 30 minutes at 4° C. in 200 μl buffer B with 5% glycerol (samples containing ψ contained a final concentration of 0.5M urea in the 200 μl as explained above). Samples were layered onto 12.3 ml gradients of 10%-30% glycerol in 25 mM Tris-HCl (pH 7.5), 0.1M NaCl and 1 mM EDTA. Protein standards in 200 HI of buffer B with 5% glycerol were layered onto another gradient and the gradients were centrifuged at 270,000×g for 44 hours at 4° C. Fractions were collected and analyzed.

The polymerase chain reaction was used to precisely clone the holC gene into the T7 based pET expression system [see Methods in Enzymology 185:60 (1990)]. Primers upstream and downstream of holC were synthesized to amplify a 604 bp fragment containing the holC gene from E. coli genomic DNA. The upstream primer hybridized over the start codon of holC and included two mismatched nucleotides in order to create an NdeI restriction site at the initiating ATG. The primer downstream of holC included three mismatched nucleotides to create a BamHI restriction site. The amplified 604 bp fragment was digested with NdeI and BamHI and cloned into the NdeI-BamHI site of the T7 based expression vector pET3c to yield pET-χ. In the pET-χplasmid, the holC gene is under the control of a strong T7 RNA polymerase promoter and an efficient Shine-Dalgarno sequence in favorable context for translation initiation. DNA sequencing of the pET-χ plasmid showed its sequence was identical to that of pUC-χ, and therefore no errors were incurred during amplification.

The pET-χ expression plasmid was introduced into strain BL21(DE)plyS which is a lysogen carrying the T7 RNA polymerase gene under the control of the IPTG-inducible lac UV5 promoter. Upon induction with IPTG and continued growth for 3 hours, the χ protein was expressed to a level of 27% total cell protein. Upon cell lysis, only about 30% of the χ protein was in the soluble fraction, the rest being found in the cell debris. Induction at lower temperature (20° C.) or for shorter times did not appear to increase the proportion of χ in the soluble fraction.

Four liters of induced cells were lysed and 38 mg of pure χ was obtained in 38% overall yield upon fractionation with ammonium sulfate precipitation, followed by column chromatography using Q sepharose and heparin agarose. The χ protein was well behaved throughout the purification and showed no tendency to aggregate. The N-terminal sequence analysis of the pure cloned χ matched that of the holC gene indicating that the protein had been successfully cloned and purified. The expressed c protein also comigrated with the authentic χ subunit contained within polIII*.

In summary, as a result of the present invention, the location and sequence of χ was determined. The χ subunit (400 pmol) was separated from γ and χ subunits of the γχψ complex by SDS denaturation and resolution on a 15% polyacrylamide gel, and 100 pmol transferred to a PVDF immobilon membrane for amino terminal sequence analysis; the remainder was transferred to nitrocellulose for sequence analysis of internal peptides following trypsin digestion. After transfer, the protein was visualized by Ponceau S stain and excised from the gel. The sequence of the N-terminal amino acids and four internal peptides were determined as described above, and these sequences were used to search the GenBank database. One single exact match was found at about 96.5 minutes on the E. coli chromosome between the xerB and valS genes.

The recombinant Kohara λ clone 5C4, contains the DNA fragment encompassing the xerB and partial valS genes, and the χ gene was subcloned by ligation of the BamHI fragment of ξ 5C4 into pUC18. Sequence analysis was performed directly on the plasmid. As shown above, the open reading frame of the χ gene was 441 nucleotides long. Its initiation codon is 160 nucleotides downstream of the stop codon of the xerB gene, while its termination codon, TAA, has one base overlapping with the start codon of the valS gene. Since the xerB and χ genes were transcribed in the same direction, and that no promoter consensus sequences were found for the χ gene alone, suggests that these two genes are in the same operon.

When PC R was applied to clone the χ gene into the T7 based expression system, PCR primers based upon the known sequences of the xerB and valS genes were made to amplify the fragment between the two genes. As described, E. coli genomic DNA was used as the PCR template, and a fragment of approximately 600 base pairs was amplified. The PCR fragment, after being digested with NdeI and BamHI, was cloned into the NdeI-BamHI site of the expression vector pET3c in similar manner to what was done with the preceding gene sequences. Thus, the putative χ gene was put under the control of a strong T7 RNA polymerase promoter gene as well as the efficient translation initiation signal, and transcription termination sequence downstream of the BamHI site. Direct DNA sequencing of the plasmids formed showed that they were all identical to the sequence of the λ clone.

The resulting plasmids were transformed into E. coli BL21(DE)pLysS that contained a lysogen carrying the T7 RNA polymerase gene under the control of the IPTG-inducible lac UV5 promoter [see Methods in Enzymology, 185:60 (1990)]. Transformants were selected by ampicillin and chloramphenicol resistance, and subsequently subjected to IPTG induction as described above. A protein of about 17 kDa was overproduced in all three PCR clones. The γ complex was run in parallel with the three clones on SDS-PAG gel, and when the overproduced and the χ subunit were at similar amounts, they showed the same gel mobility. This observation supported the identity of the overproduced protein as the χ subunit.

In addition to the specific sequences provided above for the individual genes according to the present invention, the present invention also extends to mutations, deletions and additions to these sequences provided such changes do not substantially affect the present properties of the listed sequences.

As described, the naturally occurring holenzyme consists of 10 protein subunits and is capable of extending DNA faster than polymerase I, and producing a product many times larger then the polymerase I enzyme. Thus, these unique properties of the 5, preferably 6, active subunits of the present invention are likely to find wide application in, for example, long chain PCR--using the active sequence according to the present invention PCR can be performed over several tens of kb; PCR performed at room temperature--the active sequence according to the present invention is uniquely adapted to be a polymerase of choice for PCR at room temperature due to its high fidelity; extension of site mutated primers without catalyzing strand displacement; and for sequencing operations wherein other polymerases find difficulty. Other uses will become more apparent to those skilled in the art as the science of molecular genetics continues to progress.

The sequence listing for the nucleic acid sequences and peptide sequences which are contained in the present description is as follows:

Thus, while I have illustrated and described the preferred embodiment of my invention, it is to be understood that this invention is capable of variation and modification, and I therefore do not wish to be limited to the precise terms set forth, but desire to avail myself of such changes and alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 60                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:28 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       MetLeuArgLeuTyrProGluGlnLeuArgAlaGlnLeuAsnGlu                                  51015                                                                          GlyLeuArgAlaAlaTyrLeuLeuLeuGlyAsnAspPro                                        2025                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:21 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       AlaAlaTyrLeuLeuLeuGlyAsnAspProLeuLeuLeuGln                                     510                                                                            GluSerGlnAspAlaValArg                                                          1520                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:14 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       AlaGlnGluAsnAlaAlaTrpPheThrAlaLeuAlaAsnArg                                     510                                                                            (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:24 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       ValGluGlnAlaValAsnAspAlaAlaHisPheThrProPheHis                                  51015                                                                          TrpValAspAlaLeuLeuMetGlyLys                                                    20                                                                             (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:33 base pairs                                                       (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       GTACAACCGAATCATATGTTACCCAGCGAGCTC33                                            (2) INFORMATION FOR SEQ ID NO: 6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1032 bp                                                             (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       ATGATTCGGTTGTACCCGGAACAACTCCGCGCGCAGCTC39                                      AATGAAGGGCTGCGCGCGGCGTATCTTTTACTTGGTAAC78                                      GATCCTCTGTTATTGCAGGAAAGCCAGGACGCTGTTCGT117                                     CAGGTAGCTGCGGCACAAGGATTCGAAGAACACCACACT156                                     TTTTCCATTGATCCCAACACTGACTGGAATGCGATCTTT195                                     TCGTTATGCCAGGCTATGAGTCTGTTTGCCAGTCGACAA234                                     ACGCTATTGCTGTTGTTACCAGAAAACGGACCGAATGCG273                                     GCGATCAATGAGCAACTTCTCACACTCACCGGACTTCTG312                                     CATGACGACCTGCTGTTGATCGTCCGCGGTAATAAATTA351                                     AGCAAAGCGCAAGAAAATGCCGCCTGGTTTACTGCGCTT390                                     GCGAATCGCAGCGTGCAGGTGACCTGTCAGACACCGGAG429                                     CAGGCTCAGCTTCCCCGCTGGGTTGCTGCGCGCGCAAAA468                                     CAGCTCAACTTAGAACTGGATGACGCGGCAAATCAGGTG507                                     CTCTGCTACTGTTATGAAGGTAACCTGCTGGCGCTGGCT546                                     CAGGCACTGGAGCGTTTATCGCTGCTCTGGCCAGACGGC585                                     AAATTGACATTACCGCGCGTTGAACAGGCGGTGAATGAT624                                     GCCGCGCATTTCACCCCTTTTCATTGGGTTGATGCTTTG663                                     TTGATGGGAAAAAGTAAGCGCGCATTGCATATTCTTCAG702                                     CAACTGCGTCTGGAAGGCAGCGAACCGGTTATTTTGTTG741                                     CGCACATTACAACGTGAACTGTTGTTACTGGTTAACCTG780                                     AAACGCCAGTCTGCCCATACGCCACTGCGTGCGTTGTTT819                                     GATAAGCATCGGGTATGGCAGAACCGCCGGGGCATGATG858                                     GGCGAGGCGTTAAATCGCTTAAGTCAGACGCAGTTACGT897                                     CAGGCCGTGCAACTCCTGACACGAACGGAACTCACCCTC936                                     AAACAAGATTACGGTCAGTCAGTGTGGGCAGAGCTGGAA975                                     GGGTTATCTCTTCTGTTGTGCCATAAACCCCTGGCGGAC1014                                    GTATTTATCGACGGTTGA1032                                                         (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:127 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       CCGAACAGCTGATTCGTAAGCTGCCAAGCATCCGTGCTGCGGATATTCGT50                           TCCGACGAAGAACAGACGTCGACCACAACGGATACTCCGGCAACGCCTGC100                          ACGCGTCTCCACCACGCTGGGTAACTG127                                                 (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:102 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       TATGAAATCTTTACAGGCTCTGTTTGGCGGCACCTTTGATCCGGTGCACT50                           ATGGTCATCTAAAACCCGTTGGAAGCGTGGCCGAAGTTTTGATTGGTCTG100                          AC102                                                                          (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:343 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       MetIleArgLeuTyrProGluGlnLeuArgAlaGlnLeuAsnGlu                                  51015                                                                          GlyLeuArgAlaAlaTyrLeuLeuLeuGlyAsnAspProLeuLeu                                  202530                                                                         LeuGlnGluSerGlnAspAlaValArgGlnValAlaAlaAlaGln                                  354045                                                                         GlyPheGluGluHisHisThrPheSerIleAspProAsnThrAsp                                  505560                                                                         TrpAsnAlaIlePheSerLeuCysGlnAlaMetSerLeuPheAla                                  657075                                                                         SerArgGlnThrLeuLeuLeuLeuLeuProGluAsnGlyProAsn                                  808590                                                                         AlaAlaIleAsnGluGlnLeuLeuThrLeuThrGlyLeuLeuHis                                  95100105                                                                       AspAspLeuLeuLeuIleValArgGlyAsnLysLeuSerLysAla                                  110115120                                                                      GlnGluAsnAlaAlaTrpPheThrAlaLeuAlaAsnArgSerVal                                  125130135                                                                      GlnValThrCysGlnThrProGluGlnAlaGlnLeuProArgTrp                                  140145150                                                                      ValAlaAlaArgAlaLysGlnLeuAsnLeuGluLeuAspAspAla                                  155160165                                                                      AlaAsnGlnValLeuCysTyrCysTyrGluGlyAsnLeuLeuAsn                                  170175180                                                                      LeuAlaGlnAlaLeuGluArgLeuSerLeuLeuTrpProAspGly                                  185190195                                                                      LysLeuThrLeuProArgValGluGlnAlaValAsnAspAlaAla                                  200205210                                                                      HisPheThrProPheHisTrpValAspAlaLeuLeuMetGlyLys                                  215220225                                                                      SerLysArgAlaLeuHisIleLeuGlnGlnLeuArgLeuGlyGly                                  230235240                                                                      SerGluProValIleLeuLeuArgThrLeuGlnArgGluLeuLeu                                  245250255                                                                      LeuLeuValAsnLeuLysArgGlnSerAlaHisThrProLeuArg                                  260265270                                                                      AlaLeuPheAspLysHisArgValTrpGlnAsnArgArgGlyMet                                  275280285                                                                      MetGlyGluAlaLeuAsnArgLeuSerGlnThrGlnLeuArgGln                                  290295300                                                                      AlaValGlnLeuLeuThrArgThrGluLeuThrLeuLysGlnAsp                                  305310315                                                                      TyrGlyGlnSerValTrpAlaGluLeuGluGlyLeuSerLeuLeu                                  320325330                                                                      LeuCysHisLysProLeuAlaAspValPheIleAspGly                                        335340343                                                                      (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:334 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      MetArgTrpTyrProTrpLeuArgProAspPheGluLysLeuVal                                  51015                                                                          AlaSerTyrGlnAlaGlyArgGlyHisHisAlaLeuLeuIleGln                                  202530                                                                         AlaLeuProGlyMetGlyAspAspAlaLeuIleTyrAlaLeuSer                                  354045                                                                         ArgTyrLeuLeuCysGlnGlnProGlnGlyHislysSerCysGly                                  505560                                                                         HisCysArgGlyCysGlnLeuMetGlnAlaGlyThrHisProAsp                                  657075                                                                         TyrTyrThrLeuAlaProGluLysGlyLysAsnThrLeuGlyVal                                  808590                                                                         AspAlaValArgGluValThrGluLysLeuAsnGluHisAlaArg                                  95100105                                                                       LeuGlyGlyAlaLysValValTrpValThrAspAlaAlaLeuLeu                                  110115120                                                                      ThrAspAlaAlaAlaAsnAlaLeuLeuLysThrLeuGluGluPro                                  125130135                                                                      ProAlaGluThrTrpPhePheLeuAlaThrArgGluProGluArg                                  140145150                                                                      LeuLeuAlaThrLeuArgSerArgCysArgLeuHisTyrLeuAla                                  155160165                                                                      ProProProGluGlnTyrAlaValThrTrpLeuSerArgGluVal                                  170175180                                                                      ThrMetSerGlnAspAlaLeuLeuAlaAlaLeuArgLeuSerAla                                  185190195                                                                      GlySerProGlyAlaAlaLeuAlaLeuPheGlnGlyAspAsnTrp                                  200205210                                                                      GlnAlaArgGluThrLeuCysGlnAlaLeuAlaTyrSerValPro                                  215220225                                                                      SerGlyAspTrpTyrSerLeuLeuAlaAlaLeuAsnHisGluGln                                  230235240                                                                      AlaProAlaArgLeuHisTrpLeuAlaThrLeuLeuMetAspAla                                  245250255                                                                      LeuLysArgHisHisGlyAlaAlaGlnValThrAsnValAspVal                                  260265270                                                                      ProGlyLeuValAlaGluLeuAlaAsnHisLeuSerProSerArg                                  275280285                                                                      LeuGlnAlaIleLeuGlyAspValCysHisIleArgGluGlnLeu                                  290295300                                                                      MetSerValThrGlyIleAsnArgGluLeuLeuIleThrAspLeu                                  305310315                                                                      LeuLeuArgIleGluHisTyrLeuGlnProGlyValValLeuPro                                  320325330                                                                      ValProHisLeu                                                                   (2) INFORMATION FOR SEQ ID NO: 11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 nucleic acids                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      ACTCTGGAAGAACCGCCGGCTGAAACTTGGTTTTTTCTGGCTACTCGTGA50                           ACCGGAA57                                                                      (2) INFORMATION FOR SEQ ID NO: 12:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 54 nucleic acids                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                      GCTGGTTCTCCGGGTGCTGCTCTGGCTCTGTTTCAGGGTGATGACTGGCA50                           GGCT54                                                                         (2) INFORMATION FOR SEQ ID NO: 13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1002 bp                                                             (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      ATGAGATGGTATCCATGGTTACGACCTGATTTCGAAAAA39                                      CTGGTAGCCAGCTATCAGGCCGGAAGAGGTCACCATGCG78                                      CTACTCATTCAGGCGTTACCGGGCATGGGCGATGATGCT117                                     TTAATCTACGCCCTGAGCCGTTATTTACTCTGCCAACAA156                                     CCGCAGGGCCACAAAAGTTGCGGTCACTGTCGTGGATGT195                                     CAGTTGATGCAGGCTGGCACGCATCCCGATTACTACACC234                                     CTGGCTCCCGAAAAAGGAAAAAATACGCTGGGCGTTGAT273                                     GCGGTACGTGAGGTCACCGAAAAGCTGAATGAGCACGCA312                                     CGCTTAGGTGGTGCGAAAGTCGTTTGGGTAACCGATGCT351                                     GCCTTACTAACCGACGCCGCGGCTAACGCATTGCTGAAA390                                     ACGCTTGAAGAGCCACCAGCAGAAACTTGGTTTTTCCTG429                                     GCTACCCGCGAGCCTGAACGTTTACTGGCAACATTACGT468                                     AGTCGTTGTCGGTTACATTACCTTGCGCCGCCGCCGGAA507                                     CAGTACGCCGTGACCTGGCTTTCACGCGAAGTGACAATG546                                     TCACAGGATGCATTACTTGCCGCATTGCGCTTAAGCGCC585                                     GGTTCGCCTGGCGCGGCACTGGCGTTGTTTCAGGGAGAT624                                     AACTGGCAGGCTCGTGAAACATTGTGTCAGGCGTTGGCA663                                     TATAGCGTGCCATCGGGCGATTGGTATTCGCTGCTAGCG702                                     GCCCTTAATCATGAACAAGTCCCGGCGCGTTTACACTGG741                                     CTGGCAACGTTGCTGATGGATGCGCTAAAACGCCATCAT780                                     GGTGCTGCGCAGGTGACCAATGTTGATGTGCCGGGCCTG819                                     GTCGCCGAACTGGCAAACCATCTTTCTCCCTCGCGCCTG858                                     CAGGCTATACTGGGGGATGTTTGCCACATTCGTGAACAG897                                     TTAATGTCTGTTACAGGCATCAACCGCGAGCTTCTCATC936                                     ACCGATCTTTTACTGCGTATTGAGCATTACCTGCAACCG975                                     GGCGTTGTGCTACCGGTTCCTCATCTT1002                                                (2) INFORMATION FOR SEQ ID NO: 14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:157 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      AAGAATCTTTCGATTTCTTTAATCGCACCCGCGCCCGCTATCTGGAACTG50                           GCAGCACAAGATAAAAGCATTCATACCATTGATGCCACCCAGCCGCTGGA100                          GGCCGTGATGGATGCAATCCGCACTACCGTGACCCACTGGGTGAAGGAGT150                          TGGACGC157                                                                     (2) INFORMATION FOR SEQ ID NO: 15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:143 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      TTAGAGAGACATCATGTTTTTAGTGGACTCACACTGCCATCTC43                                  GATGGTCTGGATTATGAATCTTTGCATAAGGACGTGGATGACGTTCTGGC93                           GAAAGCCGCCGCACGCGATGTGAAATTTTGTCTGGCAGTCGCCACAACAT143                          (2) INFORMATION FOR SEQ ID NO: 16:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:16 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      MetArgTrpTyrProProLeuArgProAspPheGluLysLeuValAla                               51015                                                                          (2) INFORMATION FOR SEQ ID NO: 17:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:11 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                      GluValThrGluLysLeuAsnGluHisAlaArg                                              510                                                                            (2) INFORMATION FOR SEQ ID NO: 18:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:20 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                      ValValTrpValThrAspAlaAlaLeuLeuThrAspAlaAlaAla                                  51015                                                                          AsnAlaLeuLeuLys                                                                20                                                                             (2) INFORMATION FOR SEQ ID NO: 19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:25 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                      ThrLeuGluGluProProAlaGluThrTrpPhePheLeuAlaThr                                  51015                                                                          ArgGluProGluArgLeuLeuAlaThrLeu                                                 2025                                                                           (2) INFORMATION FOR SEQ ID NO: 20:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:18 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                      LeuHisTyrLeuAlaProProProGluGlnTyrAlaValThrTrp                                  51015                                                                          LeuSerArg                                                                      18                                                                             (2) INFORMATION FOR SEQ ID NO: 21:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:21 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                      LeuSerAlaGlySerProGlyAlaAlaLeuAlaLeuPheGlnGly                                  51015                                                                          AspAsnTrpGlnAlaArg                                                             20                                                                             (2) INFORMATION FOR SEQ ID NO: 22:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:5 amino acids                                                       (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      LeuGlyGlyAlaLys                                                                (2) INFORMATION FOR SEQ ID NO: 23:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:58 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                      AlaCysThrCysThrGlyGlyAlaAlaGlyAlaAlaCysCysGly                                  51015                                                                          CysCysGlyGlyCysThrThrGlyAlaAlaAlaCysThrThrGly                                  102530                                                                         GlyThrThrThrThrThrThrCysThrGlyGlyCysThrAlaCys                                  354045                                                                         ThrCysGlyThrGlyAlaAlaCysCysGlyGlyAlaAla                                        5055                                                                           (2) INFORMATION FOR SEQ ID NO: 24:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:54 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                      GlyCysThrGlyGlyThrThrCysThrCysCysGlyGlyGlyThr                                  51015                                                                          GlyCysThrGlyCysThrCysThrGlyGlyCysThrCysThrGly                                  202530                                                                         ThrThrThrCysAlaGlyGlyGlyThrGlyAlaThrAlaAlaCys                                  354045                                                                         ThrGlyGlyCysAlaGlyGlyCysThr                                                    50                                                                             (2) INFORMATION FOR SEQ ID NO: 25:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:33 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                      GlyGlyThrGlyAlaAlaGlyGlyAlaGlyThrThrGlyGlyAla                                  51015                                                                          CysAlaThrAlaThrGlyAlaGlyAlaThrGlyGlyThrAlaThr                                  202530                                                                         CysCysAla                                                                      (2) INFORMATION FOR SEQ ID NO: 26:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:40 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                      MetLeuLysAsnLeuAlaLysLeuAspGlnThrGluMetAspLys                                  51015                                                                          ValAsnValAspLeuAlaAlaAlaGlyValAlaPheLysGluArg                                  202530                                                                         TyrAsnMetProValIleAlaGluAlaVal                                                 3540                                                                           (2) INFORMATION FOR SEQ ID NO: 27:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:57 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                      ATGCTGAAAAACCTGGCTAAACTGGATCAGACTGAAATGGATAAAGTTAA50                           CGTTGAT57                                                                      (2) INFORMATION FOR SEQ ID NO: 28:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:57 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                      CTGGCTGCTGCTGGTGTTGCTTTTAAGGAACGTTATAACATGCCGGTTAT50                           TGCTGAA57                                                                      (2) INFORMATION FOR SEQ ID NO: 29:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:228 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                      ATGCTGAAGAATCTGGCTAAACTGGATCAAACAGAAATG39                                      GATAAAGTGAATGTCGATTTGGCGGCGGCCGGGGTGGCA78                                      TTTAAAGAACGCTACAATATGCCGGTGATCGCTGAAGCG117                                     GTTGAACGTGAACAGCCTGAACATTTGCGCAGCTGGTTT156                                     CGCGAGCGGCTTATTGCCCACCGTTTGGCTTCGGTCAAT195                                     CTGTCACGTTTACCTTACGAGCCCAAACTTAAA228                                           (2) INFORMATION FOR SEQ ID NO: 30:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:172 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                      AGGCGTAGCGAAGGGAGCGTGCAGTTGAAGCCATATTATCTA42                                   TTCCTTTTTGTAATAACTTTTTTACAGACGATAACCTTGTCTAATGTCTG92                           AGTCGAGGATCATCAATTCCGGCTTGCCATCCTGGCTCACTCTTAGTAAC142                          TTTTGCCCGCGAATGATGAGGAGATTAAGA172                                              (2) INFORMATION FOR SEQ ID NO: 31:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:107 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                      TAAAACTTATACAGAGTTACACTTTCTTACATAACGCCTGCT42                                   AAATTATGAGTATTTTCTAAACCGCACTCATAATTTGCAGTCATTTTGAA92                           AAGGAAGTCATTATG107                                                             (2) INFORMATION FOR SEQ ID NO: 32:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:76 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                      MetLeuLysAsnLeuAlaLysLeuAspGlnThrGluMetAspLys                                  51015                                                                          ValAsnValAspLeuAlaAlaAlaGlyValAlaPheLysGluArg                                  202530                                                                         TyrAsnMetProValIleAlaGluAlaValGluArgGluGlnPro                                  354045                                                                         GluHisLeuArgSerTrpPheArgGluArgLeuIleAlaHisArg                                  505560                                                                         LeuAlaSerValAsnLeuSerArgLeuProTyrGluProLysLeu                                  657075                                                                         Lys                                                                            76                                                                             (2) INFORMATION FOR SEQ ID NO: 33:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:40 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                      MetLeuLysAsnLeuAlaLysLeuAspGlnThrGluMetAspLys                                  51015                                                                          ValAsnValAspLeuAlaAlaAlaGlyValAlaPheLysGluAla                                  202530                                                                         TyrAsnMetProValIleAlaGluAlaVal                                                 3540                                                                           (2) INFORMATION FOR SEQ ID NO: 34:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:57 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                      ATGCTGAAAAACCTGGCTAAACTGGATCAGACTGAAATGGAT42                                   AAAGTTAACGTTGAT57                                                              (2) INFORMATION FOR SEQ ID NO: 35:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:57 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                      CTGGCTGCTGCTGGTGTTGCTTTTAAAGAACGTTATAACATG42                                   CCGGTTATTGCTGAA57                                                              (2) INFORMATION FOR SEQ ID NO: 36:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:33 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                      ATGATGAGGAGATTACATATGCTGAAGAATCTG33                                            (2) INFORMATION FOR SEQ ID NO: 37:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:51 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                      GAGGAATTCGGCTTTTTTGCCGAATTCCTCGGCCCCTAGGAGATCTCAGC50                           T51                                                                            (2) INFORMATION FOR SEQ ID NO: 38:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:137 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                      MetThrSerArgArgAspTrpGlnLeuGlnGlnLeuGlyIleThr                                  51015                                                                          GlnTrpSerLeuArgArgProGlyAlaLeuGlnGlyGluIleAla                                  202530                                                                         IleAlaIleProAlaHisValArgLeuValMetValAlaAsnAsp                                  354045                                                                         LeuProAlaLeuThrAspProLeuValSerAspValLeuArgAla                                  505560                                                                         LeuThrValSerProAspGlnValLeuGlnLeuThrProGluLys                                  657075                                                                         IleAlaMetLeuProGlnGlySerHisCysAsnSerTrpArgLeu                                  808590                                                                         GlyThrAspGluProLeuSerLeuGluGlyAlaGlnValAlaSer                                  95100105                                                                       ProAlaLeuThrAspLeuArgAlaAsnProThrAlaArgAlaAla                                  110115120                                                                      LeuTrpGlnGlnIleCysThrTyrGluHisAspPhePheProGly                                  125130135                                                                      AsnAsp                                                                         137                                                                            (2) INFORMATION FOR SEQ ID NO: 39:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:411 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                      ATGACATCCCGACGAGACTGGCAGTTACAGCAACTGGGC39                                      ATTACCCAGTGGTCGCTGCGTCGCCCTGGCGCGTTGCAG78                                      GGCGAGATTGCCATTGCGATCCCGGCACACGTCCGTCTG117                                     GTGATGGTGGCAAACGATCTTCCCGCCCTGACTGATCCT156                                     TTAGTGAGCGATGTTCTGCGCGCATTAACCGTCAGCCCC195                                     GACCAGGTGCTGCAACTGACGCCAGAAAAAATCGCGATG234                                     CTGCCGCAAGGCAGTCACTGCAACAGTTGGCGGTTGGGT273                                     ACTGACGAACCGCTATCACTGGAAGGCGCTCAGGTGGCA312                                     TCACCGGCGCTCACCGATTTACGGGCAAACCCAACGGCA351                                     CGCGCCGCGTTATGGCAACAAATTTGCACATATGAACAC390                                     GATTTCTTCCCTGGAAACGAC411                                                       (2) INFORMATION FOR SEQ ID NO: 40:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:77 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                      GGCGATTATAGCCATATGTTGGCGCGGTATCGACGAATTTGCTATATTTG50                           CGCCCCTGACAACAGGAGCGATTCGCT77                                                  (2) INFORMATION FOR SEQ ID NO: 41:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:103 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                      TGATTTACCGGCAGCTTACCACATTGAACAACGCGCCCACGCC43                                  TTTCCGTGGAGTGAAAAAACGTTTGCCAGCAACCAGGGCGAGCGTTATCT93                           CAACTTTCAG103                                                                  (2) INFORMATION FOR SEQ ID NO: 42:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:27 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                      GATTCCATATGACATCCCGACGAGACT27                                                  (2) INFORMATION FOR SEQ ID NO: 43:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:30 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                      GACTGGATCCCTGCAGGCCGGTGAATGAGT30                                               (2) INFORMATION FOR SEQ ID NO: 44:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:17 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                      LeuGlyThrAspGluProLeuSerLeuGluGluAlaGlnValAla                                  51015                                                                          SerPro                                                                         17                                                                             (2) INFORMATION FOR SEQ ID NO: 45:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:17 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                      AlaAlaLeuTrpGlnGlnIleCysThrTyrGluHisAspPhePhe                                  51015                                                                          ProAla                                                                         17                                                                             (2) INFORMATION FOR SEQ ID NO: 46:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:32 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                      CAACAGGAGCGATTCCATATGACATCCCGACG32                                             (2) INFORMATION FOR SEQ ID NO: 47:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:31 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                      GATTCGGATCCCTGCAGGCCGGTGAATGAGT31                                              (2) INFORMATION FOR SEQ ID NO: 48:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:30 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                      CCCCACATATGAAAAACGCGACGTTCTACC30                                               (2) INFORMATION FOR SEQ ID NO: 49:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:28 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                      ACCCGGATCCAAACTGCCGGTGACATTC28                                                 (2) INFORMATION FOR SEQ ID NO: 50:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:441 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                      ATGAAAAACGCGACGTTCTACCTTCTGGACAATGACACC39                                      ACCGTCGATGGCTTAAGCGCCGTTGAGCACCTGGTGTGT78                                      GAAATTGCCGCAGAACGTTGGCGCAGCGGTAAGCGCGTG117                                     CTCATCGCCTGTGAAGATGAAAAGCAGGCTTACGCCCTG156                                     GATGAAGCCCTGTGGGCGCGTCCGGCAGAAAGCTTTGTT195                                     CCGCATAATTTAGCGGGAGAAGGACCGCGCGGCGGTGTA234                                     CCGGTGGAGATCGCCTGGCCGCAAAAGCGTAGCAGCAGC273                                     CGGCGCGATATATTGATTAGTCTGCGAACAAGCTTTGCA312                                     GATTTTGCCACCGCTTTTACAGAAGTGGTAGACTTCGTT351                                     CCTCATGAAGATTCTCTGAAACAACTGGCGCGCGAACGC390                                     TATAAAGCCTACCGCGTGGCTGGTTTCAACCTGAATACG429                                     GCAACTTGGAAA441                                                                (2) INFORMATION FOR SEQ ID NO: 51:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:175 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                      TAACGGCGAAGAGTAATTGCGTCAGGCAAGGCTGTTATTGCCGGATGCGG50                           CGTGAACGCCTTATCCGACCTACACAGCACTGAACTCGTAGGCCTGATAA100                          GACACAACAGCGTCGCATCAGGCGCTGCGGTGTATACCTGATGCGTATTT150                          AAATCCACCACAAGAAGCCCCATTT175                                                   (2) INFORMATION FOR SEQ ID NO: 52:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:100 bp                                                              (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                      TAATGGAAAAGACATATAACCCACAAGATATCGAACAGCCGCTTTACGAG50                           CACTGGGAAAAAAGCCAGGAAAGTTTCTGCATCATGATCCCGCCGCCGAA100                          (2) INFORMATION FOR SEQ ID NO: 53:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:147 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                      MetLysAspAlaThrPheTyrLeuLeuAspAsnAspThrThrVal                                  51015                                                                          AspGlyLeuSerAlaValGluGlnLeuValCysGluIleAlaAla                                  202530                                                                         GluArgTrpArgSerGlyLysArgValLeuIleAlaCysGluAsp                                  354045                                                                         GluLysGlnAlaTyrArgLeuAspGluAlaLeuTrpAlaArgPro                                  505560                                                                         AlaGluSerPheValProHisAsnLeuAlaGlyGluGlyProArg                                  657075                                                                         GlyGlyAlaProValGluIleAlaTrpProGlnLysArgSerSer                                  808590                                                                         SerArgArgAspIleLeuIleSerLeuArgThrSerPheAlaAsp                                  95100105                                                                       PheAlaThrAlaPheThrGluValValAspPheValProTyrGlu                                  110115120                                                                      AspSerLeuLysGlnLeuAlaArgGluArgTyrLysAlaTyrArg                                  125130135                                                                      ValAlaGlyPheAsnLeuAsnThrAlaThrTrpLys                                           140145147                                                                      (2) INFORMATION FOR SEQ ID NO: 54:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:29 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                      MetLysAsnAlaThrPheTyrLeuLeuAspAsnAspThrThrVal                                  51015                                                                          AspGlyLeuSerAlaValGluGlnLeuValXaaGluIleAla                                     2025                                                                           (2) INFORMATION FOR SEQ ID NO: 55:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:9 amino acids                                                       (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                      ValLeuIleAlaXaaGluAspGluLys                                                    5                                                                              (2) INFORMATION FOR SEQ ID NO: 56:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:21 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                      LeuAspGluAlaLeuTrpAlaAlaProAlaGluSerPheValPro                                  51015                                                                          HisAsnLeuAlaGlyGlu                                                             20                                                                             (2) INFORMATION FOR SEQ ID NO: 57:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:10 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                      GlyGlyAlaProValGluIleAlaTrpPro                                                 510                                                                            (2) INFORMATION FOR SEQ ID NO: 58:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:8 amino acids                                                       (B) TYPE: amino acid                                                           (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                      GlyPheAsnLeuAsnThrAlaThr                                                       5                                                                              (2) INFORMATION FOR SEQ ID NO: 59:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:30 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                      CCCCACATATGAAAAACGCGACGTTCTACC30                                               (2) INFORMATION FOR SEQ ID NO: 60:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:28 bp                                                               (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                      ACCCGGATCCAAACTGCCGGTGACGTTC28                                                 __________________________________________________________________________

Having thus described my invention and the manner and a process of making and using it in such full, clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same; 

I claim:
 1. An isolated conformationally correct protein subunit of polymerase III holoenzyme from Escherichia coli selected from the subunit group consisting of θ and χ.
 2. The isolated protein according to claim 1, wherein the subunit group is θ.
 3. The isolated protein according to claim 2, wherein the protein has a molecular weight of 8.6 kDa.
 4. The isolated protein according to claim 2, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 32. 5. The isolated protein according to claim 1, wherein the subunit is χ.
 6. The isolated protein according to claim 5, wherein the protein has a molecular weight of 17 kDa.
 7. The isolated protein according to claim 5, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 53. 8. The isolated protein according to claim 5, wherein the protein is purified.
 9. The isolated protein according to claim 5, wherein the protein is recombinant.
 10. An isolated conformationally correct ψ protein subunit of polymerase III holenzyme from Escherichia coli, wherein the protein subunit is segregated from other proteins.
 11. An isolated DNA molecule encoding a ψ protein subunit of polymerase III holenzyme from Escherichia coli which is segregated from other proteins.
 12. The isolated protein according to claim 10, wherein the protein has a molecular weight of 15.2 kDa.
 13. The isolated protein according to claim 10, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 38. 14. The isolated DNA molecule according to claim 11, wherein the subunit has a molecular weight of 15.2 kDa.
 15. The isolated DNA molecule according to claim 11, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 38. 16. An expression system comprising a DNA molecule according to claim 11 in a vector heterologous to the DNA molecule.
 17. An isolated conformationally correct protein δ subunit of polymerase III holenzyme having an amino acid sequence corresponding to SEQ. ID. No.
 9. 18. An isolated conformationally correct protein δ' subunit of polymerase III holenzyme having an amino acid sequence corresponding to SEQ. ID. No.
 10. 19. An isolated DNA molecule encoding a protein subunit of polymerase III holenzyme from Escherichia coli wherein the subunit group is θ.
 20. An isolated DNA molecule encoding a protein subunit of polymerase III holoenzyme from Escherichia coli, wherein the subunit group is θ.
 21. The isolated DNA molecule according to claim 20, wherein the subunit has a molecular weight of 8.6 kDa.
 22. The isolated DNA molecule according to claim 20, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 32. 23. The isolated DNA molecule according to claim 22, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 29. 24. The isolated DNA molecule according to claim 15, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 39. 25. An isolated DNA molecule encoding a protein subunit of polymerase III holenzyme from Escherichia coli, wherein the subunit is χ.
 26. The isolated DNA molecule according to claim 25, wherein the subunit has a molecular weight of 17 kDa.
 27. The isolated DNA molecule according to claim 25, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 53. 28. The isolated DNA molecule according to claim 27, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 50. 29. The isolated DNA molecule according to claim 19, wherein the subunit has a molecular weight of 38.7 kDa.
 30. The isolated DNA molecule according to claim 19, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 9. 31. The isolated DNA molecule according to claim 30, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 6. 32. An isolated DNA molecule encoding a protein subunit of polymerase III holenzyme from Escherichia coli, wherein the subunit group is δ'.
 33. The isolated DNA molecule according to claim 32, wherein the subunit has a molecular weight of 36.9 kDa.
 34. The isolated DNA molecule according to claim 32, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 10. 35. The isolated DNA molecule according to claim 34, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 13. 36. An expression system comprising a DNA molecule according to claim 20 in a vector heterologous to the DNA molecule.
 37. The expression system according to claim 36, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 32. 38. The expression system according to claim 37, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 29. 39. The expression system according to claim 16, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 38. 40. The expression system according to claim 39, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 39. 41. An expression system comprising a DNA molecule according to claim 25 in a vector heterologous to the DNA molecule.
 42. The expression system according to claim 41, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 53. 43. The expression system according to claim 42, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 50. 44. An expression system comprising a DNA molecule according to claim 19 in a vector heterologous to the DNA molecule.
 45. The expression system according to claim 44, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 9. 46. The expression system according to claim 45, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 6. 47. An expression system comprising a DNA molecule according to claim 32 in a vector heterologous to the DNA molecule.
 48. The expression system according to claim 47, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 10. 49. The expression system according to claim 48, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 13. 50. A host cell transformed with a heterologous DNA molecule according to claim
 19. 51. The host cell according to claim 50, wherein the DNA molecule is inserted into a heterologous expression system.
 52. A host cell transformed with a heterologous DNA molecule according to claim
 20. 53. The host cell according to claim 52, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 32. 54. The host cell according to claim 53, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 29. 55. A host cell transformed with a heterologous DNA molecule according to claim
 11. 56. The host cell according to claim 55, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 38. 57. The host cell according to claim 56, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 39. 58. A host cell transformed with a heterologous DNA molecule according to claim
 25. 59. The host cell according to claim 58, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 53. 60. The host cell according to claim 59, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 50. 61. The host cell according to claim 50, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 9. 62. The host cell according to claim 61, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 6. 63. A host cell transformed with a heterologous DNA molecule according to claim
 32. 64. The host cell according to claim 63, wherein the protein has an amino acid sequence corresponding to SEQ. ID. No.
 10. 65. The host cell according to claim 64, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ. ID. No.
 13. 66. The host cell according to claim 55, wherein the DNA molecule is inserted into a heterologous expression system.
 67. The host cell according to claim 58, wherein the DNA molecule is inserted into a heterologous expression system.
 68. The host cell according to claim 63, wherein the DNA molecule is inserted into a heterologous expression system.
 69. The isolated protein δ subunit according to claim 17, wherein the protein is purified.
 70. The isolated protein δ subunit according to claim 17, wherein the protein is recombinant.
 71. The isolated protein δ' subunit according to claim 18, wherein the protein is purified.
 72. The isolated protein δ' subunit according to claim 18, wherein the protein is recombinant.
 73. The isolated protein subunit according to claim 12, wherein the protein is purified.
 74. The isolated protein subunit according to claim 12, wherein the protein is recombinant.
 75. The isolated protein subunit according to claim 2, wherein the protein is purified.
 76. The isolated protein subunit according to claim 2, wherein the protein is recombinant. 